WO2013007003A1 - Novel use of acetylacetic acid for regulating proliferation of skeletal-muscle cells - Google Patents

Abstract

Disclosed is the use of acetylacetic acid in preparing drugs for promoting the proliferation of skeletal-muscle cells, the skeletal-muscle cells being myoblasts, wherein the acetylacetic acid promotes the proliferation of the skeletal-muscle cells by activating the ERK pathway so as to up-regulate the expression of a cyclin, such as cyclin D1. Also disclosed is the use of acetylacetic acid in preparing drugs for treating injuries to skeletal muscle, and a drug composition for promoting the proliferation of skeletal-muscle cells, containing acetylacetic acid and Insulin-like Growth Factor 1.

Description

 New use of acetoacetate to regulate skeletal muscle cell proliferation

 The present invention relates to the regulation of skeletal muscle cell cycle regulation and cell proliferation, and more particularly to the novel use of acetoacetate in the regulation of cell cycle-associated proteins and the regulation of skeletal muscle cell proliferation. The invention further relates to the use of acetoacetate in the manufacture of a medicament for the treatment of skeletal muscle damage. Background technique

 The long-term research on cell proliferation mostly focuses on the regulation of cell growth by growth factors and other cytokines. The uptake of nutrients by cells is also regulated by growth factors, and some growth factor signaling pathways and their transcriptional levels have been discovered. Adjust the network.

 Insulin-like growth factor 1 (IGF1) is similar in structure and function to insulin, and plays an important role in normal body growth and cell proliferation, differentiation, and metabolism. IGF is produced in the liver and exerts biological effects through circulation to various tissues of the body. Skeletal muscle is an important target organ. IGF1 plays an important role in the development and maintenance of skeletal muscle. Mice deficient in the IGF1 receptor develop severe muscle atrophy and die shortly after birth due to the inability to perform normal lung respiration. In contrast, skeletal muscle volume in mice overexpressing IGF1 is significantly increased

[1]. At the same time, in the muscle hypertrophy model, such as the muscle hypertrophy model caused by exercise, the expression of IGF1 is significantly increased [2].

IGF1 binds to its receptor and stimulates the Ras-raf-MEK-ERK signaling pathway and PI3K-Akt signaling pathway. These two pathways are essential for cell proliferation and survival. Among them, most of the studies related to IGF1 are the Akt pathway. Studies have shown that the activation of PI3K is essential for skeletal muscle hypertrophy [3]. PI3K then activates a series of proteins such as downstream Akt, mTOR, up-regulates transcriptional regulators involved in protein translation, and promotes protein synthesis by increasing the initiation and elongation of translation, leading to skeletal muscle hypertrophy.

 MSTN is a member of the TGF-β superfamily and is highly conserved among different species, mainly expressed in skeletal muscle [4], and also expressed in non-skeletal muscle tissues such as glands and myocardium [5, 6] . The skeletal muscle of MSTN knockout mice is three times that of wild-type mice, indicating that MSTN has an inhibitory effect on skeletal muscle growth. The double-muscle cattle with MSTN gene mutation and abnormal skeletal muscle also provide the same evidence [7]. . At the same time, the use of in situ hybridization to study the expression of MSTN during mouse embryonic development also suggests that MSTN also plays a crucial role in skeletal muscle development [4].

MSTN regulates the molecular mechanism of cell proliferation, and found that MSTN can up-regulate the cell cycle inhibitor p21, thereby inhibiting the activity of CDK2, and inhibiting the transcriptional activity of E2F by Rb protein, ultimately blocking the cell from G1 to S phase [8] ]. In addition, some studies have found that MSTN can also inhibit the conversion of cells from G2 to M phase through p21 [9]. Our laboratory study provides another pathway for the mechanism of action of MSTN, ie, MSTN can down-regulate the expression of cyclinD1 through the PI3K-Akt-GSK3P pathway, thereby arresting the cell cycle in G1 phase, and finding that this effect can interact with IGF1. In order to antagonize, it is proposed that MSTN may cooperate with IGF1 to regulate the role of cell proliferation [10]. In addition, both in vivo and in vitro studies have shown that MSTN can also inhibit the proliferation of satellite cells by up-regulating the expression of p21, suggesting an important role for MSTN in skeletal muscle regeneration [11]. There are not many studies on endogenous metabolites and cell proliferation in the prior art, and no unified theoretical model has been proposed. But more and more studies have shown that metabolites such as cholesterol and certain unsaturated fatty acids can regulate cell proliferation. In addition, the concept of endogenous metabolite lactic acid, a very important signal molecule in sugar metabolism, has recently been proposed [12]. Recent studies have found that lactic acid plays a role in wound healing, suggesting that it has independent biological functions outside the metabolic process.

 The endogenous metabolite acetoacetate is a downstream metabolite of beta-hydroxybutyrate (β-ΗΒ). As a major component of the ketone body, acetoacetate and β-hydroxybutyric acid are synthesized in the liver into the circulatory system, and catabolism in the extrahepatic tissue produces sputum, which provides energy to the body. Especially in the case of long-term starvation, ketone bodies can be used as a substitute for sugar by the tissues of the brain, heart, kidneys and skeletal muscles to maintain the life activities of the body. It has been reported in the literature that the metabolite β-ΗΒ in the renal cell line regulates cell proliferation, whereas acetoacetate does not have this effect; the opposite result is found in endothelial cells [13]. The effects of acetoacetate and β-indole may have specificity in different tissue cells. Summary of the invention

 A first aspect of the invention provides a novel use of acetoacetate in the regulation of cell cycle associated proteins. The present inventors have found that acetoacetate has a regulatory effect on the cyclin Dl. In particular, the inventors have found that acetoacetate can upregulate the expression level of cyclinDl at the transcriptional and translational levels. Meanwhile, the inventors have demonstrated that acetoacetate up-regulates the expression of cyclin by activating the ERK pathway.

A second aspect of the invention provides a novel acetoacetate for promoting skeletal muscle cell proliferation Use. In particular, the skeletal muscle cells are myoblasts. The inventors have demonstrated that acetoacetate promotes myoblast proliferation by increasing the ratio of s in a dose- and time-dependent manner. Specifically, the inventors demonstrated that the endogenous metabolite acetoacetate upregulates the expression of the cyclin D1 by activating the ERK pathway, thereby promoting the proliferation of myoblasts.

 In a third aspect, the invention provides a pharmaceutical composition for promoting skeletal muscle cell proliferation comprising acetoacetate and insulin-like growth factor 1, and a pharmaceutically acceptable carrier. The present inventors have found that acetoacetic acid can synergize with insulin-like growth factor (IGF1) or antagonize the regulation of cell cycle by MSTN.

 In a fourth aspect, the invention further provides a method of treating skeletal muscle damage comprising administering an effective amount of acetoacetate to an individual in need thereof. Furthermore, the present invention provides a method of treating skeletal muscle damage comprising administering to a subject in need thereof a pharmaceutical composition comprising acetoacetate and insulin-like growth factor 1, and a pharmaceutically acceptable carrier.

 In another aspect, the invention further provides the use of acetoacetate in the manufacture of a medicament for treating skeletal muscle damage. In particular, the skeletal muscle damage is induced by cyclic phosphate.

 The invention will be more clearly understood in conjunction with the following embodiments and with reference to the drawings. DRAWINGS

Fig. 1A, IB, 1C, ID. Acetoacetic acid (AA) can promote the proliferation of C ₂ C ₁₂ cells, while the effect of β-ΗΒ is not obvious (**: ρ < 0.01).

Figure 2. Acetoacetic acid (ΑΑ) promotes C ₂ C ₁₂ cell proliferation in a dose-dependent manner (**: p<0.01) o

 Figure 3. Acetoacetic acid (AA) promotes C2C12 cell proliferation in a time-dependent manner (**: p < 0.01, p < 0.001).

 Figure 4. Acetoacetic acid (AA) upregulates the expression of cyclinDl.

 Figure 5. Acetoacetic acid (AA) activates the ERK pathway (* * : p < 0.01, ***: p < 0.001) o

Figure 6. Acetoacetic acid (AA) promotes proliferation of C ₂ C ₁₂ cells by activating the ERK pathway (*: p<0.05, **: p<0.01) o

 Figure 7. Acetoacetic acid (AA) synergizes with IGF1 to promote cell proliferation or antagonizes MSTN's ability to inhibit cells (*: p<0.05, **: p<0.01) o

 Figure 8. Acetoacetic acid (AA) significantly attenuates cyclic phosphate (CTX damage to skeletal muscle tissue.

 1. Cell culture

(3⁄4 ( ₁₂ cells are mouse myoblast cell lines established by Yaffe and Saxel. The cells exhibit fibroblast morphology under normal growth conditions, under appropriate conditions (eg, high cell density or serum starvation) C ₂ C ₁₂ cells can differentiate into contractile myotubes and express specific muscle differentiation marker proteins. Treatment of the cells with bone morphogenic protein 2 (BMP-2) can result in their muscle formation. Transformation of cells into osteoblasts. The culture conditions of C ₂ C ₁₂ cells in the proliferation stage are Dulbecco's modified Eagle's medium (DMEM) medium containing 10% fetal calf serum. The antibiotic content was 100 Units/mL penicillin and 100 g/mL streptomycin, and the growth group was growth medium (GM). The culture environment of the cells was 37 ° C and contained 5% C0 ₂ .

It should be noted that: (^ ₂ ( ₁₂ cells are strictly prohibited from growing at a density of 100% confluent during culture. Therefore, when the cell density reaches about 60%, digestive juice is required (0.25% Trypsin, 0.53 mM). EDTA) is passaged at a ratio of 1: 2 or 1: 3.

 Cell recovery

The cells stored in liquid nitrogen were taken out and quickly placed in a 37 ° C water bath to melt. After centrifugation at 1000 rpm for 5 min, discard the supernatant and collect the cells. Add 10% fetal bovine serum to the complete medium, gently pipe the cells, transfer to a 25 cm ² flask, and add 5 mL of complete medium, 37 ° C, 5% C0. ₂ Incubate in the incubator.

 Cryopreservation of cells

 Fresh medium was exchanged overnight before cryopreservation of cells. The cells were collected by trypsinization at the time of cryopreservation. Add appropriate amount of frozen solution (containing 10% dimethyl sulfoxide, 20% fetal bovine serum in complete medium), resuspend the cells, transfer to a cryotube, freeze at -70 ° C overnight and transfer to liquid nitrogen. save.

2. Acetoacetic acid promotes the proliferation of myoblast C ₂ C ₁₂

First, we examined the effects of acetoacetate (purchased from Sigma-Aldrich, catalog no. A8509) and β-ΗΒ (purchased from Sigma-Aldrich, catalog no. 54965) on the proliferation of myoblast C ₂ C ₁₂ . Myoblast C ₂ C ₁₂ was treated with 5 mM acetoacetic acid and 5 mM β-purine, respectively, and the cells were collected for flow and MTT detection after 48 hours. Streaming The results showed (Fig. 1A, B, C) that acetoacetate significantly increased the proportion of cells in the S phase, ie acetoacetate promoted the transition of cells from G1 to S phase, but the endogenous metabolite β-ΗΒ had no effect on the cell cycle. (where IGF1 is used as a positive control). The sputum test also yielded results consistent with the flow pattern. Acetoacetic acid significantly increased cell viability, whereas β-ΗΒ had no significant effect on cell viability (Fig. 1D).

To determine whether acetoacetate promotes the proliferation of myoblast C ₂ C ₁₂ in a dose-dependent manner, we treated C ₂ C ₁₂ cells with O.lmM, 0.5 mM, 1 mM, 5 mM acetoacetate for 48 hours. The collected cells were separately subjected to H ³ TdR and flow detection. The results of H ³ TdR showed that the DNA synthesis of the cells increased with the increase of the dose of acetoacetate. When the dose was increased to above 1 mM, the increase of DNA synthesis was statistically significantly different from that of the control group. That is, acetoacetate can promote DNA synthesis of C ₂ C ₁₂ cells in a dose-dependent manner ( FIG. 2A ). At the same time, the flow results showed that with the increase of the dose of acetoacetate, the proportion of S phase in the cells gradually increased. When the dose was increased to 5 mM, the increase of S phase was statistically significant compared with the control group. That is, acetoacetate can promote the conversion of C ₂ C ₁₂ cells from the G1 phase to the S phase in a dose-dependent manner (Fig. 2B). The above results indicate that acetoacetate promotes C ₂ C ₁₂ cell proliferation in a dose-dependent manner.

According to the above dose test, we treated C ₂ C ₁₂ cells with 5 mM acetoacetate, collected cells at 24 hours, 36 hours, and 48 hours, and examined the time dependence of acetoacetate to promote cell proliferation. The H ³ TdR results showed that the DNA synthesis of the cells gradually increased with the prolongation of the treatment time. There was a significant difference in the increase in DNA synthesis between the 24 hours of treatment and the control group (p<0.01). After 36 hours of treatment, the increase in DNA synthesis was significantly different from the control group (p<0.001). That is, acetoacetate can be time dependent The relict mode promoted DNA synthesis of C ₂ C ₁₂ cells (Fig. 3A). At the same time, the results of flow analysis showed that the proportion of S phase in cells treated for 24 hours did not change significantly. However, with the prolongation of treatment time, the proportion of S phase in cells increased gradually, and the S phase of cells treated for 48 hours increased. There was a significant difference compared to the control group. It was shown that acetoacetate promoted the conversion of C ₂ C ₁₂ cells from G1 phase to S phase in a time-dependent manner (Fig. 3B). In summary, acetoacetate promotes C ₂ C ₁₂ cell proliferation in a time-dependent manner.

3. Acetoacetic acid promotes cell proliferation by up-regulating cyclinD1 expression

The above results indicate that acetoacetate promotes C ₂ C ₁₂ cell proliferation by increasing the ratio of S phase in a dose- and time-dependent manner. Changes in the cell cycle are regulated by changes in cyclin expression. Therefore, we have further studied how acetoacetate promotes the proliferation of C ₂ C ₁₂ cells by affecting which cell cycle-associated proteins. First, we treated the proliferation with 5 mM acetoacetate ( ₂ ( ₁₂ cells, cells were collected after 48 hours, and the expression of cell cycle-associated proteins was detected by Western blot. The results showed that the expression of cyclinDl in the cells treated with acetoacetate compared with the control group. There is a significant up-regulation, and the expression of cyclinE is also up-regulated (Fig. 4A). Since cyclinDl and cyclinE mainly regulate the cell transition from G1 to S phase, this is consistent with the results of the above cell cycle analysis. The acetylacetate treatment of C ₂ C ₁₂ cells detected the expression of cyclinD1 at the RNA level and protein level. Realtime-PCR and Western blot results showed that acetoacetate promoted cyclinDl expression in a dose-dependent manner at the RNA and protein levels. (Fig. 4B, C). 4. Acetoacetic acid promotes proliferation of C ₂ C ₁₂ cells by activating ERK pathway

Acetoacetic acid can promote the proliferation of C ₂ C ₁₂ cells by up-regulating the expression of cyclinD1. According to the relationship between the known MAPK signaling pathway and cell cycle regulation, ERK can affect the cell cycle by regulating the expression of cyclinDl. Therefore, we first examined the effect of related proteins on the ERK pathway on acetoacetate regulation of C ₂ C ₁₂ cell proliferation. The cells at different times were collected for 5 mM acetoacetate for protein level detection. The results of Western blotting showed that the phosphorylated ERK protein level gradually increased with the treatment time (Fig. 5A, 5B). To further confirm that ERK is involved in the signaling process of acetoacetate, we examined changes in the upstream proteins p-MEKl/2 and pc-Raf in the ERK signaling pathway. The results showed that the protein levels of phosphorylated MEK1/2 and c-Raf also showed a gradual increase with the prolongation of acetoacetate treatment time (Fig. 5A, 5C, 5D). This indicates that acetoacetate activates the raf-MEK-ERK pathway.

Although acetoacetate activates the raf-MEK-ERK pathway, is this signaling pathway directly involved in the regulation of the cell cycle by acetoacetate? To answer this question, C ₂ C ₁₂ inhibitor PD98059 we ERK pathway to block ERK pathway, while expression was detected in cell proliferation and cyclinDl. The H ³ TdR results showed that PD98059 alone treated cells inhibited DNA synthesis compared to the control, indicating that the inhibitor was effective. Treatment of cells with acetoacetate alone can promote DNA synthesis. However, when acetoacetate was treated in combination with the inhibitor, the synthesis of DNA was decreased compared to the acetoacetate alone treatment group, and this decrease was more pronounced as the inhibitor concentration increased (Fig. 6A). This suggests that activation of the ERK pathway is involved in the regulation of acetaminoacetate to promote cell proliferation. At the same time, we examined the expression changes of cyclinDl. Western blot results show that PD98059 single Treatment of cells alone can reduce the level of phosphorylation of ERK, and treatment of cells with acetoacetate alone can increase the level of phosphorylated ERK protein. The combination of acetoacetate and inhibitors resulted in a decrease in phosphorylated ERK protein levels compared to treatment with only acetoacetate, and this decreasing trend was more pronounced with increasing PD98059 dose (Figure 6B, 6C). The trend of expression of CyclinDl was consistent with the trend of p-ERK (Fig. 6B, 6D). The above results indicate that the endogenous metabolite acetoacetate up-regulates the expression of cyclinD1 by activating the ERK pathway, thereby promoting the proliferation of C ₂ C ₁₂ cells.

5. The effect of acetoacetate on the proliferation of C ₂ C ₁₂ cells in combination with IGF1 or antagonizing MSTN

Proliferating C ₂ C ₁₂ cells were treated with acetoacetate (5 mM), IGF1 (50 ng/ml), and MSTN (500 ng/ml), respectively or in combination. Flow cytometry was consistent with previous results. Treatment with acetoacetate and IGF1 alone increased the proportion of S phase in cells, while treatment with MSTN alone reduced the proportion of S phase. Significantly, the combination of acetoacetate and IGF1 increased the S phase of cells treated with IGF1 alone, indicating that acetoacetate can synergize with IGF1 to promote cell proliferation. In contrast, treatment with acetoacetate in combination with MSTN restored the reduced S phase of MSTN treatment to control levels, suggesting that acetoacetate can antagonize the inhibition of cell cycle by MSTN (Fig. 7A). Further treatment with different doses of acetoacetate in combination with IGF1 or MSTN showed that acetoacetate synergistically synergized with IGF1 or antagonized MSTN on the C ₂ C ₁₂ cell cycle in a dose-dependent manner ( FIG. 7B ).

It is known that IGF1 or MSTN can regulate the cell cycle through the ERK signaling pathway. To further determine whether acetoacetate and IGF1 or MSTN work together to regulate cell proliferation through the ERK pathway, we interfered with the cell cycle with the PD98059 inhibitor of the ERK pathway. Flow cytometry results showed that PD98059 only partially inhibited cell cycle progression compared to the control group (Fig. 7C). This result suggests that acetoacetate and IGF1 or MSTN can work together to regulate C ₂ C ₁₂ cell proliferation through the ERK pathway.

 6. Acetoacetic acid can significantly attenuate the damage of CTX to skeletal muscle tissue In order to detect the biological function of acetoacetate in animals, we used the CTX-induced skeletal muscle injury model. CTX, different doses of acetoacetate were injected alone or in combination with 8-week-old C57BL/6 mouse gastrocnemius muscle. Tissue samples were taken for histological and molecular biological tests 4 days after injection. Tissue section staining and Western Blot results showed that CTX alone caused extensive damage to skeletal muscle tissue, and a large number of immune cell infiltrates were observed under the microscope (Fig. 8A). Western Blot results showed that the expression of muscle satellite cell activation Marker increased. Injection of acetoacetate alone had little damage to skeletal muscle tissue (Fig. 8B). Simultaneous injection of CTX with acetoacetate significantly reduced skeletal muscle damage compared with CTX alone, and the expression of muscle satellite cell activation Marker was also significantly decreased (Fig. 8A, 8B). The above results show that acetoacetate can significantly reduce the damage of CTX to skeletal muscle tissue. references

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Claims

Claims:  1. Use of acetoacetate for the preparation of a medicament for promoting skeletal muscle cell proliferation.  2. The use according to claim 1, wherein the skeletal muscle cells are myoblasts.  3. The use according to claim 1, wherein acetoacetic acid promotes proliferation of skeletal muscle cells by up-regulating expression of a cell cycle protein.  4. The use according to claim 3, wherein the cyclin is cyclinDl.  5. The use according to claim 3, wherein acetoacetate upregulates cyclin expression by activating the ERK pathway.  6. Use of acetoacetate in the preparation of a medicament for the treatment of skeletal muscle damage.  7. The use according to claim 6, wherein the skeletal muscle damage is induced by cyclophosphamide.  A pharmaceutical composition for promoting skeletal muscle cell proliferation comprising acetoacetic acid and insulin-like growth factor 1, and a pharmaceutically acceptable carrier.  9. A method of treating skeletal muscle damage comprising administering an effective amount of acetoacetate to an individual in need thereof.  10. A method of treating skeletal muscle damage comprising administering to a subject in need thereof an effective amount of acetoacetate and insulin-like growth factor 1.