Abstract
Disclosed is a composition for promoting local muscle growth or slowing down or preventing local muscle atrophy, which composition contains a polypeptide in the C2 region of the enterotoxin Staphylococcus aureus and a myostatin polypeptide. By means of the composition, the defect in the prior art of only systemic muscle growth being possible has been overcome so as to achieve effects of promoting local muscle growth, or slowing down or preventing local muscle atrophy.
Claims
1. A composition for promoting local muscle growth or slowing local muscle atrophy, wherein the composition comprises a first polypeptide; a second polypeptide; and a linker between the first peptide and the second peptide, wherein the sequence of the first polypeptide, the linkerand the second polypeptide issetforth in SEQ ID NO: 17.
2. A method for promoting local muscle growth or slowing local muscle atrophy comprising a step of administeringto a subject in a local muscle in need thereof the effective amount of the composition as claimed in claim 1.
3. The method according to claim 2, wherein the composition is used for muscle atrophy caused by nerve trauma.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The present disclosure will become more fully understood from the detailed description and the accompanying drawings, in which:
(2) FIG. 1 is a line graph related to the ratio of muscle cross-sectional area of a low-dose experimental group and a low-dose control group of the present invention. The low-dose experimental group is indicated by a broken line indicating .circle-solid. and the low-dose control group was indicated by a broken line indicating .square-solid..
(3) FIG. 2 is a line graph related to the ratio of the muscle cross-sectional area of medium dose experimental group and a medium-dose control group of the present invention. The medium-dose experimental group is indicated by a broken line indicating .circle-solid., and the medium-dose control group is indicated by a broken line indicating .square-solid..
(4) FIG. 3 is a line graph related to the ratio of the muscle cross-sectional area of the high-dose experimental group of the present invention compared with the high-dose control group. The high-dose experimental group is indicated by a broken line indicating .circle-solid., and the high-dose control group is indicated by a broken line indicating .square-solid..
(5) FIG. 4 is a longitudinal sectional view and a cross-sectional scan of the computerized tomography of the mouse hind leg of the present invention, each cross-sectional scan is a cross section of the cross-sectional line of the corresponding longitudinal section scan; FIG. 4 (A) is a longitudinal section scan of the low-dose control group, and the muscle volume is calculated by the area between the upper and lower dashed lines; FIG. 4 (B) is a longitudinal section scan of the medium-dose control group, and the muscle volume is calculated by the area between the upper and lower dashed lines; FIG. 4 (C) is a longitudinal section scan of the high-dose control group, and the muscle volume is calculated by the area between the upper and lower dashed lines; FIG. 4 (D) is a cross sectional scan of the low-dose control group; FIG. 4 (E) is a cross sectional scan of the medium-dose control group; FIG. 4 (F) is a cross sectional scan of the high-dose control group; FIG. 4 (G) is a longitudinal section scan of the low-dose experimental group, and the muscle volume is calculated by the area between the upper and lower dashed lines; FIG. 4 (H) is a longitudinal section scan of the medium-dose experimental group, and the muscle volume is calculated by the area between the upper and lower dashed lines; FIG. 4 (I) is a longitudinal section scan of the high-dose experimental group, and the muscle volume is calculated by the area between the upper and lower dashed lines; FIG. 4 (J) is a cross sectional scans of the low-dose experimental group; FIG. 4 (K) is a cross sectional scan of the medium-dose experimental group; FIG. 4 (L) is a cross sectional scan of a high-dose experiment group.
(6) FIG. 5 is a bar graph of the volume ratio of the mice hind leg of the low-dose control group and the low-dose experimental group of the present invention; A.U. indicates an arbitrary unit.
(7) FIG. 6 is a bar graph of the volume ratio of the mice hind leg of the medium-dose control group and the medium-dose experimental group of the present invention; A.U. indicates an arbitrary unit.
(8) FIG. 7 is a bar graph of the volume ratio of the mice hind leg of the high-dose control group and the high-dose experimental group of the present invention; A.U. indicates an arbitrary unit.
(9) FIG. 8 is a staining diagram of hematoxylin-eosin (H-E) tissue staining of the mice calf muscle fibers of the high-dose control group of the present invention.
(10) FIG. 9 is a staining diagram of the H-E tissue staining of the mice calf muscle fibers of the high-dose experimental group of the present invention.
(11) FIG. 10 is a diagram of immunohistochemical staining of mice calf muscle fibers administered myostatin of the present invention; wherein FIGS. 10 (A) to 10 (C) are control groups; FIGS. 10 (D) to 10(F) are respectively 50 ng (low-dose), 500 ng (medium-dose) and 5000 ng (high-dose) experimental group.
(12) FIG. 11 is a line graph related to the weight change after administering of the composition in low-dose, medium-dose and high-dose experimental group of the present invention. wherein the low-dose experimental group is indicated by a broken line indicating .circle-solid., the medium-dose experimental group is indicated by a broken line indicating .square-solid., and the high-dose experimental group is indicated by a broken line indicating .box-tangle-solidup..
(13) FIG. 12 is a bar graph related to the ratio of muscle volume after damaging sciatic nerve and then administered 1000 ng composition of the present invention in low-dose, medium-dose and high-dose experimental group of the present invention; wherein the sciatic nerve of the left leg was injured, but the sciatic nerve of right leg was not damaged.
(14) FIG. 13 is a bar graph related to the muscle ratio obtained from the left leg muscle volume ratio divided by the right leg muscle volume ratio in the control group, and that in the experimental group of FIG. 12.
(15) FIG. 14 is a bar graph related to the ratio of muscle volume after blocking sciatic nerve and then administered 1000 ng or 5000 ng composition of the present invention in experimental group A and experimental B of the present invention; wherein the sciatic nerve of the left leg was blocked, but the sciatic nerve of right leg was not blocked.
(16) FIG. 15 is a bar graph related to the ratio of muscles obtained from the left leg muscle volume ratio divided by the right leg muscle volume ratio in the experimental group A and experimental B of FIG. 14.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
(17) The technical means adopted by the present invention for achieving the intended purpose of the invention are further described below in conjunction with the drawings and preferred embodiments of the invention.
Preparation Example 1
Preparation of the Composition Comprising an SEC2 Fragment and a Myostatin Fragment
(18) The fusion protein used in this embodiment was pET expression system of Escherichia coli; preferably, the expression system was pET-28a. Wherein the first polypeptide “SEC2m” was a SEC2 having a point mutation, the nucleic acid sequence was set forth in SEQ ID NO: 1, and the protein sequence was set forth in SEQ ID NO: 8, wherein the point mutation was: 7T>L, 9G>E, 13Y>V, and 105H>Y. The second polypeptide “Myo epitope” was epitope of myostatin, which was 15 amino acids with 6 repeats in C-terminal and highly conserved in multiple species (as set forth in SEQ ID NO: 14). the nucleic acid sequence of a single fragment was set forth in SEQ ID NO :2. The gene sequence located in the multiple cloning site (MCS) of the pET vector from the N-terminus was SEC2m, linker and Myo epitope, which was set forth in SEQ ID NO: 3.
(19) 35 L fermentation culture process was established, to culture Escherichia coli BL21(DE3) strain containing the fragment set forth in SEQ ID NO: 3 in a 50 L fermentation tank 4 tubes of 5 mL bacterial strain cultured overnight with LB/Ampicillin medium at 37° C. were respectively inoculated into 0.2 L LB/Ampicillin medium and a total of 1 L were shook and cultured at 37° C. to OD600 of 0.3, and then added into 35 L medium for culturing, and sampled for determining OD600 every two hours to monitor the variations of the growth curve. Suitable time points were further selected according to the growth curve, IPTG having a final concentration of 0.1 mM was added to induce Escherichia coli to express the fusion protein at a high level, shook and cultured at 37° C. for 3 hours, and centrifuged to recover the bacteria. The expression of the fusion protein was determined through SDS-PAGE and western blotting, to determine the optimum 35 L fermentation conditions. Generally, the fusion polypeptide expressed by Escherichia coli BL21 (DE3) was subjected to extraction and isolation of the fusion polypeptide after cell lysis, and then obtaining a composition set forth in SEQ ID NO: 17. The extraction and isolation were conventional techniques.
Example 1
Promoting Local Muscle Growth Test
(20) The mice used in the animal experiments of this example were 8 week old (12 month old) female mice of the C57BL/6 strain, and a total of 9 mice in the control group and the experimental group. The period of experiment was 6 months. When the mice grew to 12 months, they were feed with high-fat diets and raised water with high fructose syrup. Feed and water would be replaced every two days to avoid deterioration. The feed was stored at −20° C., and the high fructose syrup was stored at 4° C.; the body weight was measured every two weeks. After 6 months of feeding, the experimental animals began intramuscular injection of the composition once a week. The composition obtained from preparation Example 1 diluted into a different concentration by physiological saline was injected into the muscles of the left hind calf of the mice into different doses as the experimental group, and the right lower hind calf was injected with saline. The experimental groupings were shown in Table 1.
(21) The grouping of animal experiment and the administration method of composition of the present invention
(22) TABLE-US-00002 The dose of each The volume of Amount administered to intramuscular of Fre- Group each mouse injection animal quency Low-dose 50 ng 10 μL 3 once a week Medium-dose 500 ng 10 μL 3 once a week High-dose 5000 ng 10 μL 3 once a week
(23) The collection of experimental data was measured weekly by a vernier scale, measuring the long diameter (a), short diameter (b) and body weight of the muscle; the measurement position was the position of the injection. The approximately cross-sectional area was calculated as the ellipse area: a×b×3.14, and the muscle volume was calculated by the area between the upper and lower dashed lines in FIG. 4 (A), FIG. 4 (B), FIG. 4 (C), FIG. 4 (G), FIG. 4 (H), FIG. 4 (I).
(24) (1) Comparing the muscle cross-sectional area of the experimental group (left hind calf) and the control group (right hind calf) after injecting the composition of the present invention
(25) As shown in FIGS. 1 to 3, each experimental group was compared with the control group at the week 19, wherein the cross-sectional area of the calf injected with the high-dose composition was increased by 8.19% compared with the high-dose control group. The cross-sectional area of the calf with the medium-dose composition increased by 5.5% compared to the medium-dose control group. The cross-sectional area of the low-dose composition was increased by 5.67% compared to the low-dose control group.
(26) (2) Comparing the muscle volume of the experimental group (left hind calf) and the control group (right hind calf) after injecting the composition of the present invention
(27) Referring to FIG. 4, a longitudinal cross-sectional scan and a cross-sectional scan of the computerized tomography of the mouse hind calf, each cross-sectional scan is a cross-section of the cross-sectional line of the corresponding longitudinal section scan. the range of the muscle volume was calculated by the area between the dashed lines in FIG. 4 (A), FIG. 4 (B), FIG. 4 (C), FIG. 4 (G), FIG. 4 (H), FIG. 4 (I), wherein the muscle volume was obtained by integrating each cross-sectional area. The experiment was terminated after administration until 19 weeks. The mice were sacrificed and their calves were fixed with 10% formalin, and then subjected to computed tomography.
(28) Referring to FIGS. 5 to 7, the muscle volume of the low-dose experimental group increased by 4.6% compared with the low-dose control group; the muscle volume of the medium-dose experimental group increased by 8.5% compared with the middle-dose control group; the muscle volume of the high-dose experimental group increased by 19.2% compared with the high-dose control group. Since the test was based on the muscle of the left hind calf as the experimental group, and the right hind calf as the control group in an identical mouse, the results showed that the injection of the composition of the present invention into the left calf only causes the left calf muscle to grow on the side. On the other side (right side), the muscles of the calf did not grow, so that the composition of the present invention produced muscle growth only by the local administration, but did not cause systemic muscle enlargement.
(29) (3) Measuring the thickness of the myofilament fiber after the injection in the experimental group (left hind calf) and the control group (right hind calf)
(30) At week 19, the calf bones of the mice were taken out after scanning computerized tomography and sacrificing, and then the muscles were embedded in paraffin and sectioned, and the thickness of the myofilament fibers was compared by H-E tissue staining. Referring to FIG. 8 and FIG. 9, FIG. 8 was the muscle fiber of the hind calf of the high-dose group as the control group, and FIG. 9 was the muscle fiber of the hind calf of the high-dose group as the experimental group. The muscle fiber of the high-dose group (the experimental group) was significantly increased compared to the control group.
(31) (4) Measurement of the distribution of myostatin content after injection of the experimental group (left hind calf) and the control group (right hind calf)
(32) At week 19, the calf bones of the mice were taken out after scanning computerized tomography and sacrificing, and then the muscles were embedded in paraffin and sectioned, and the distribution of myostatin content was observed with myostatin antibody by immunohistochemical staining (IHC). Referring to FIG. 10, myostatin can be significantly stained in the different doses of the control groups (left posterior calf) as shown in FIG. 10 (A), FIG. 10 (B), and FIG. 10 (C). In contrast, the expressions of myostatin were significantly inhibited in the low dose, the medium dose, or the high dose experimental group (right hind calf) as shown in FIG. 10 (D), FIG. 10 (E), and FIG. 10 (F). That is, regardless of the low, the medium or the high dose group, the right hind calf (ie, the control group) had a high concentration of myostatin and the right hind calf muscle did not become significantly larger compared to the muscle of the left hind calf in an identical mouse. In other words, myostatin of the left hind calf (ie, the experimental group) was inhibited by the composition of the present invention, so that the myostatin concentration was low, and the muscle of the left hind calf in the identical mouse was enlarged, that is, the composition of the present invention has the effect of increasing local muscle mass, but not affected by systemic blood circulation.
(33) (5) Comparing changes in body weight after injection of the low-dose, the medium-dose or the high doses of the composition of the present invention
(34) Body weight was measured at week 19, as shown in FIG. 11, there was no significant difference in body weight either in the low-dose, the medium-dose or the high-dose group after administration of the composition of the present invention. Therefore, this test showed that the composition of the present invention only increases the muscle at the local administration site and does not increase systemic muscle.
Example 2
Slowing or Preventing Local Muscle Atrophy Caused by Nerve Damaged Test
(35) 10 ICR mice (eight weeks old, purchased from Lesco Biotech Co., Ltd.) fed in normal diet for 1 week to 2 weeks, and then divided into 2 groups (5 mice in each group). In the day I, the left leg sciatic nerve of each group of mice (no sciatic nerve injury in the right leg of each group) were undergone sciatic nerve injury surgery to caused sciatic nerve injury. The mice were sacrificed on day 28. The control group was the mice that underwent sciatic nerve injury surgery to destroy the sciatic nerve of the left leg, but did not receive any administration. The experimental group was the mice that underwent sciatic nerve injury surgery to destroy that the sciatic nerve of the left leg and each mouse in the experimental group was intramuscularly injected on days 1, 3, 7, and 14 with 1000 ng composition obtained from the preparation example 1 of the present invention. The level of muscle atrophy was observed to evaluate the effect of the composition of the present invention on slowing or preventing muscle atrophy.
(36) The sciatic nerve injury surgery was performed under abdominal anesthesia. The body hair was removed from the knee to the buttocks of the mouse, fixed the mouse's legs and disinfected the surgical site with alcohol cotton, found the position of the thigh femur and opened an incision near the buttocks parallel femur. After the muslce layer was peeled off, a sciatic nerve parallel to the femur would be seen. Picking up the sciatic nerve and injuring by special tools, and then returning the sciatic nerve to the original position and observing the wound healing, gait changes and overall status of the mouse daily after suturing the skin. The objective was designed to mimic the state of nerve injury.
(37) The results showed that on the day 28, the volume of the left thigh muscle of one of the control group mice was about 1373 mm3, and the volume of the right thigh muscle was about 1595 mm3. Because the mice did not undergo sciatic nerve injury surgery on their right thighs, so the volume of the right thigh muscle can be regarded as the baseline and the ratio is 1. The volume ratio of the left thigh muscle is the volume of the left thigh muscle divided by the volume of the right thigh muscle (as shown in the control group in FIG. 12). It showed that the muscles of the left thigh compared to the right thigh was atrophy in the control group. The volume of the left thigh muscle of one of the experimental group mice was about 1888 mm3, and the volume of the right thigh muscle was about 1705 mm3. The volume of the right thigh muscle was as the baseline ratio 1. The volume ratio of the left thigh muscle is the volume of the left thigh muscle divided by the volume of the right thigh muscle (as shown in the experimental group in FIG. 12). Although the left thigh of the mouse underwent the sciatic nerve injury surgery, the left leg muscles are not atrophied due to the administration of the composition of the present invention.
(38) As shown in FIG. 13, the left thigh/right thigh muscle volume ratio of the control group was about 0.86 (less than 1 means atrophy); the left thigh/right thigh muscle volume ratio of the experimental group was about 1.11, so the experimental group compared The control group exhibits a phenomenon of maintaining a muscle volume by administering the composition of the present invention. Thus, in the case of nerve damage, by administering the composition of the present invention, local muscle atrophy or even the maintenance of the original muscle volume can be reduced or relieved.
Example 3
Slowing or Preventing Local Muscle Atrophy Caused by Nerve Truncation
(39) 10 ICR mice (eight weeks old, purchased from Lesco Biotech Co., Ltd.) fed in normal diet for 1 week to 2 weeks, and then divided into 2 groups (5 mice in each group). In the day 1, the mice were divided into a control group, an experimental group A, and an experimental group B. The control group was the mice that underwent nerve truncation on their sciatic nerve of the left leg, but did not administer the composition of the present invention. The experimental group A was the mice that underwent nerve truncation on their sciatic nerve of the left leg, and each of them was intramuscularly injected on days 1, 3, 7, and 14 with 1000 ng composition obtained from the preparation example 1 of the present invention. The experimental group B was the mice that underwent nerve truncation on their sciatic nerve of the left leg, and each of them was intramuscularly injected on days 1, 3, 7, and 14 with 5000 ng composition obtained from the preparation example 1 of the present invention. The level of muscle atrophy was observed to evaluate the effect of the composition of the present invention against muscle atrophy. The sciatic nerve of the right leg was not performed truncation in each group. The sciatic nerve truncation procedure was similar to that of Example 2, except that the sciatic nerve was directly truncated after being picked up.
(40) The results showed on day 28, the muscle volume of the left thigh of the control group was about 1375 mm.sup.3 and the muscle volume of the right thigh was about 1560 mm.sup.3. Since the right thigh was not subjected to sciatic nerve truncation surgery, the muscle volume of the right thigh was used as the reference ratio 1. The muscle volume ratio of the left thigh was the left thigh muscle volume divided by the right thigh muscle volume (as shown in the control group in FIG. 14), indicating that the left leg muscle of the control group was atrophied compared to the right leg muscle, and the right leg muscle was not atrophied. The muscle volume of the left thigh of the experimental group A was about 1289 mm.sup.3, the muscle volume of the right thigh was about 1394 mm.sup.3, because the right leg has not undergone sciatic nerve truncation surgery, so the muscle volume of the right thigh as a reference ratio 1. The volume ratio of the left thigh muscle was that the left thigh muscle volume divided by the right thigh muscle volume (as shown in experimental group A in FIG. 14). After sciatic nerve truncation surgery, the left leg muscle atrophy was significantly slowed down. The muscle volume of the left thigh of the experimental group B mouse was about 1958 mm.sup.3, the muscle volume of the right thigh was about 1869 mm.sup.3, because the right leg was not undergone sciatic nerve truncation surgery, so the muscle volume of the right thigh as a reference ratio 1. The volume ratio of the left thigh muscle was the muscle volume of the left thigh divided by the muscle volume of the right thigh (as shown in experimental group B in FIG. 14). Although the left thigh of the mice undergoes sciatic nerve truncation surgery, the level of muscle atrophy was not observed muscle atrophy due to the administration of the composition of the present invention.
(41) As shown in FIG. 15, the muscle volume ratio of the left thigh/right thigh of the control group was about 0.87, showing atrophic state (less than 1 means that the atrophy state); the muscle volume ratio of the left thigh/right thigh of the experimental group A was about 0.92 experimental group, it showed a slowing of muscle atrophy compared with the control group. The muscle volume ratio of the left thigh/right thigh of the experimental group B was about 1.04, and showed the experimental group B could even maintain muscle volume compared with the control group. Thus, in addition the local muscle atrophy can be reduced or relieved in Example 2, the muscle volume can maintain the original state by administering the composition of the present invention in the case of the sciatic nerve truncation of the Example 3.
(42) The embodiments were chosen and described in order to explain the principles of the disclosure and their practical application so as to enable others skilled in the art to utilize the disclosure and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present disclosure pertains without departing from its spirit and scope.
