CN117257837A - Application of exosome in preparing medicine for treating muscular atrophy caused by type 2 diabetes - Google Patents
Application of exosome in preparing medicine for treating muscular atrophy caused by type 2 diabetes Download PDFInfo
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- CN117257837A CN117257837A CN202310759269.7A CN202310759269A CN117257837A CN 117257837 A CN117257837 A CN 117257837A CN 202310759269 A CN202310759269 A CN 202310759269A CN 117257837 A CN117257837 A CN 117257837A
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- exosome
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Abstract
The invention discloses an application of exosomes in preparing a medicament for treating muscular atrophy caused by type 2 diabetes mellitus, wherein the exosomes are derived exosomes of umbilical cord mesenchymal stem cells, and the exosomes improve the muscular atrophy caused by type 2 diabetes mellitus by enhancing the autophagy effect mediated by AMPK/ULK 1. The invention also provides application of the exosome in preparing medicines for treating muscular atrophy caused by obesity or disuse muscular atrophy. The invention verifies that the exosomes of the umbilical cord mesenchymal stem cells can improve the type 2 diabetes/obesity/disuse-induced muscle atrophy by enhancing the autophagy mediated by AMPK/ULK1, so the exosomes of the umbilical cord mesenchymal stem cells have good prospect in the preparation of medicaments for treating the type 2 diabetes/obesity/disuse-induced muscle atrophy.
Description
Technical Field
The invention relates to the field of biological medicine industry, in particular to application of exosomes in preparation of a medicament for treating amyotrophy caused by type 2 diabetes.
Background
Muscle atrophy refers to the decrease in muscle volume caused by the attenuation or even the disappearance of muscle fibers, and aging, braking, and some chronic diseases such as type 2 diabetes (T2 DM) and obesity all cause muscle atrophy. Type 2 diabetics often exhibit mild muscular atrophy in middle-aged years, with age and development of diabetic neuropathy becoming more severe. The causal role of obesity, diabetes and braking in muscle atrophy has been demonstrated in animal models. Muscle atrophy is caused by reduced protein synthesis and increased degradation rates. The ubiquitination-proteasome pathway and the autophagy-lysosomal pathway play an important role therein.
The stem cells have unique biological characteristics, extremely strong self-renewal capacity, multidirectional differentiation potential, secretion of various cytokines and other characteristics, and are the optimal seed cells for realizing organ tissue regeneration. The stem cell therapy can enable partial diabetics to stop insulin therapy or reduce the dosage of insulin, and the effectiveness and safety of the stem cell therapy for treating diabetes are also effectively verified. Exosomes (exosomes, EXOs) are extracellular vesicles secreted by cells containing bioactive molecules, including proteins, lipids, nucleic acids, and the like. At present, many researches at home and abroad show that the treatment based on mesenchymal stem cells (mesenchymal stem cell, MSC) plays an important role in muscle atrophy caused by various diseases.
The mesenchymal stem cells and the exosomes thereof have good application prospects in treating the muscular atrophy caused by the T2DM, however, the influence of the mesenchymal stem cells and the exosomes on the muscular atrophy of the T2DM patient is not clear.
Disclosure of Invention
The present disclosure has been made in view of the above-mentioned prior art, and an object thereof is to provide an application of exosomes in preparing a medicament for treating muscular dystrophy caused by type 2 diabetes.
To this end, a first aspect of the present disclosure provides the use of an exosome, which is a derived exosome of umbilical cord mesenchymal stem cells, for the preparation of a medicament for treating muscle atrophy caused by type 2 diabetes, the exosome improving muscle atrophy caused by type 2 diabetes by enhancing AMPK/ULK 1-mediated autophagy. In the present disclosure, it was verified that exosomes of umbilical cord mesenchymal stem cells can improve type 2 diabetes-induced muscle atrophy by enhancing AMPK/ULK 1-mediated autophagy, and therefore, exosomes of umbilical cord mesenchymal stem cells have a good prospect in the preparation of drugs for treating type 2 diabetes-induced muscle atrophy.
In the application related to the disclosure, optionally, the preparation method of the exosome includes: obtaining human umbilical cord tissue; culturing human umbilical cord tissue by using an alpha-MEM culture medium until the cell reaches 80% of fusion; culturing in exosome-free culture medium, and collecting to obtain supernatant; and centrifuging the supernatant to obtain the exosome. Thereby, umbilical cord mesenchymal stem cell-derived exosomes can be obtained by extraction from human umbilical cord tissue.
In the application of the first aspect to which the present disclosure relates, optionally, the alpha-MEM medium contains 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin. Therefore, the umbilical cord mesenchymal stem cell-derived exosomes can be prepared.
In an application of the first aspect to which the present disclosure relates, optionally, the supernatant is filtered through a 0.22 μm filter and centrifuged. Therefore, the umbilical cord mesenchymal stem cell-derived exosomes can be prepared.
In the application of the first aspect to which the present disclosure relates, optionally, the supernatant is filtered through a 0.22 μm filter and centrifuged at 120000g for 70 minutes to obtain the exosome. Therefore, the umbilical cord mesenchymal stem cell-derived exosomes can be prepared.
In an application of the first aspect to which the present disclosure relates, optionally, the exosomes increase the cross-sectional area and number of muscle fibers to improve muscle atrophy caused by type 2 diabetes.
In an application of the first aspect to which the present disclosure relates, optionally, the exosomes regulate the ratio of fast and slow muscle fibers to improve muscle atrophy caused by type 2 diabetes.
In the use of the first aspect of the present disclosure, optionally, the exosomes promote phosphorylation of AMPK and ULK1 to enhance AMPK/ULK1 mediated autophagy.
In a second aspect, the present disclosure provides a use of an exosome, which is umbilical cord mesenchymal stem cell-derived exosome, for improving muscle atrophy caused by obesity by enhancing AMPK/ULK 1-mediated autophagy, in the preparation of a medicament for treating muscle atrophy caused by obesity. In the present disclosure, it was verified that exosomes of umbilical mesenchymal stem cells can improve obesity-induced muscle atrophy by enhancing AMPK/ULK 1-mediated autophagy, and thus, exosomes of umbilical mesenchymal stem cells have a good prospect in the preparation of drugs for treating muscle atrophy caused by obesity.
In a third aspect, the disclosure provides a use of an exosome in the manufacture of a medicament for treating disuse muscle atrophy, wherein the exosome is umbilical mesenchymal stem cell-derived exosome, the exosome improving disuse muscle atrophy by enhancing AMPK/ULK 1-mediated autophagy. In the present disclosure, it was verified that exosomes of umbilical mesenchymal stem cells can improve disuse muscle atrophy by enhancing AMPK/ULK 1-mediated autophagy, and therefore, exosomes of umbilical mesenchymal stem cells have a good prospect in the application of preparing a medicament for treating disuse muscle atrophy.
According to the present disclosure, there can be provided an application of a human umbilical cord mesenchymal stem cell-derived exosome in preparing a medicament for treating muscular dystrophy caused by type 2 diabetes and/or obesity, and an application of a human umbilical cord mesenchymal stem cell-derived exosome in preparing a medicament for treating disuse muscular dystrophy.
Drawings
FIG. 1 is a graph showing the identification results of hucMSCs according to the present invention.
Fig. 2 is a graph showing the results of the test of hucMSCs of example 1 and comparative example 1 of the present invention for improving muscular atrophy caused by diabetes.
Fig. 3 is a graph showing the results of the test of hucMSCs of example 2 and comparative example 2 for relieving muscular atrophy caused by obesity.
Fig. 4 is a graph showing test results of improved brake (IM) -induced muscular atrophy of hucMSCs in example 3 and comparative example 3 of the present invention.
Fig. 5 is a graph showing the first test results of example 4 and comparative example 4 according to the present invention.
FIG. 6 is a graph showing the second test results of example 4 and comparative example 4 according to the present invention.
Fig. 7 is a graph showing the results of a test for hucMSCs of example 5 of the present invention to inhibit PA-induced myotube atrophy by enhancing AMPK/autophagy signaling pathway.
FIG. 8 is a graph showing the results of a first test of improving diabetes-induced muscle atrophy by MSC-EXO according to example 6 of the present invention.
FIG. 9 is a graph showing the second test result of improving diabetes-induced muscle atrophy by MSC-EXO according to example 6 of the present invention.
FIG. 10 is a graph showing the results of a test of MSC-EXO in example 7 of the present invention to inhibit PA-induced myotube atrophy by enhancing the AMPK/autophagy signaling pathway.
Detailed Description
Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same members are denoted by the same reference numerals, and overlapping description thereof is omitted. In addition, the drawings are schematic, and the ratio of the sizes of the components to each other, the shapes of the components, and the like may be different from actual ones.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, such as a process, method, system, article, or apparatus that comprises or has a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus, but may include or have other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In addition, headings and the like referred to in the following description of the invention are not intended to limit the scope or content of the invention, but rather are merely indicative of reading. Such subtitles are not to be understood as being used for segmenting the content of the article, nor should the content under the subtitle be limited only to the scope of the subtitle.
Proper noun abbreviations and resolution to which the present disclosure relates:
type 2 diabetes mellitus2 diabetes mellitus, T2 DM;
MSC/MSCs Mesenchymal stem cell/cells mesenchymal stem cells;
hucMSCs human umbilical cord MSCs human umbilical cord mesenchymal stem cells;
MSC-EXO, hucMC-derived exosomes derived from human umbilical cord mesenchymal stem cells;
HFD High-fat diet;
IM hindlimb immobilization hind limb braking;
PBS Phosphate-buffered saline;
IPGTT Intraperitoneal glucose tolerance test glucose tolerance test in abdominal cavity;
IPITT Intraperitoneal insulin tolerance test intraperitoneal insulin resistance test.
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements throughout or elements having like or similar functionality. The embodiments described below by way of the drawings are exemplary only and should not be construed as limiting the invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless expressly stated otherwise, as understood by those skilled in the art. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, and/or groups thereof.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
The present disclosure relates to:
application of Mesenchymal Stem Cells (MSCs) in preparing medicament for treating muscular dystrophy caused by type 2 diabetes;
use of mesenchymal stem cell-derived exosomes (MSC-EXO) for the preparation of a medicament for treating muscle atrophy caused by type 2 diabetes;
use of Mesenchymal Stem Cells (MSCs) in the manufacture of a medicament for treating muscle atrophy caused by obesity;
use of mesenchymal stem cell-derived exosomes (MSC-EXO) for the preparation of a medicament for treating muscle atrophy caused by obesity;
use of Mesenchymal Stem Cells (MSCs) in the manufacture of a medicament for the treatment of disuse muscle atrophy;
use of mesenchymal stem cell-derived exosomes (MSC-EXO) for the preparation of a medicament for disuse muscle atrophy.
In the present disclosure, in some examples, the mesenchymal stem cells may be human umbilical cord mesenchymal stem cells (hucMSCs), and the mesenchymal stem cell-derived exosomes may be human umbilical cord mesenchymal stem cell-derived exosomes. In this case, umbilical cord is used as a material source of mesenchymal stem cells and exosomes thereof, and has advantages of reduced harm to human body and easy collection when collecting compared with other materials such as bone marrow, liver, etc., and umbilical cord-derived mesenchymal stem cells have stronger proliferation and differentiation ability.
In some examples, human umbilical cord mesenchymal stem cells and their exosomes may be obtained from fresh umbilical cord tissue of a neonate.
In some examples, human umbilical cord mesenchymal stem cells may be obtained from umbilical cord tissue by culturing. In some examples, human umbilical cord tissue may be cultured using alpha-MEM medium. The medium may contain 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin. Thus, human umbilical cord mesenchymal stem cells can be obtained by culture.
In some examples, human umbilical cord mesenchymal stem cell-derived exosomes may be isolated after culturing human umbilical cord tissue using a culture medium. In some examples, the supernatant after cultivation may be filtered using a 0.22 μm filter, whereby the larger particle size component of the supernatant can be removed and the exosomes can be retained in the supernatant as much as possible. In some examples, the filtered supernatant may be centrifuged and the centrifuged precipitate is the exosome. The filtered supernatant may be centrifuged at 120000g for 70 minutes and the pellet after centrifugation is the exosome. Thus, the exosomes derived from the human umbilical mesenchymal stem cells can be prepared.
The present disclosure evaluates the efficacy of human umbilical mesenchymal stem cells and their derived exosomes for diabetes and/or obesity-induced muscle atrophy and disuse muscle atrophy, and studies the molecular mechanism of specific actions. Human umbilical cord mesenchymal stem cells or their derived exosomes have an ameliorating effect on muscular atrophy caused by diabetes and/or obesity and disuse muscular atrophy, and human umbilical cord mesenchymal stem cells or their derived exosomes ameliorate muscular atrophy by enhancing AMPK/ULK 1-mediated autophagy. In some examples, human umbilical mesenchymal stem cells or exosomes derived therefrom may increase the cross-sectional area and number of muscle fibers to improve muscle atrophy, in some examples by adjusting the ratio of fast and slow muscle fibers.
In order that the invention may be readily understood, a further description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings and are not to be construed as limiting embodiments of the invention.
It will be appreciated by those skilled in the art that the drawings are merely schematic representations of examples and that the elements of the drawings are not necessarily required to practice the invention.
Examples
In embodiments of the present disclosure, hucMSCs or hucMSCs-derived exosomes (MSC-EXO) interventions were performed on various mouse models (diabetic db/db mice, high Fat Diet (HFD) mice, hindlimb brake (IM) mice), C2C12 myotubes, and skeletal muscle strength and locomotor performance were tested using grip testing and running machine running, muscle mass was assessed using body composition, muscle weight and muscle fiber cross-sectional area (CSA), potential regulatory mechanisms were explored by RNA-seq analysis of the tibialis anterior muscle (TA) and Western blot (immunoblot) detection of related signal pathways to explore the efficacy and mechanism of hucMSCs or hucMSCs-derived exosomes (MSC-EXO) on diabetes-induced muscle atrophy.
In this example, all of the test materials used were conventional in the art and were commercially available.
The experimental method comprises the following steps:
obtaining hucMSCs and its exosomes
(1) Fresh human umbilical cord tissue was obtained from healthy newborns, after washing the umbilical cord and removing blood vessels, the Wharton's jelly was cut into small pieces and placed in a cell culture flask containing an alpha-MEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin. The medium was changed every 3 days until the cells reached 80% confluence.
(2) 3 rd to 5 th generation cells for flow cytometry analysis, differentiation induction, drug administration and Transwell TM Co-culturing. After 48 hours of culture in exosome-free medium, the cell supernatant was collected, centrifuged to remove cells and cell debris, and then filtered through a 0.22 μm sterile filter. Subsequently, the filtrate was ultracentrifuged at 120000×g at 4℃for 70 minutes, and the precipitate was an exosome. The exosome-containing pellet was resuspended in PBS and stored at-80 ℃ for subsequent experiments.
The PARTICLE size of the exosomes was detected by ZetaVIEWS/N17-310 (PARTICLE METRIX, germany). The morphology of the exosomes was detected by transmission electron microscopy. The expression of the exosome markers CD9, CD63, CD81 was detected by immunoblotting.
Establishing animal model
(1) Establishment of T2 DM-related muscle-wasting mouse model
A4-week-old male diabetic db/db mouse (Kwanes laboratory animal Co., ltd.) was obtained, fed with a normal diet, and placed in an environment with a light/dark cycle at a temperature of 22℃to 25℃and a humidity of 55.+ -. 5% for 12 hours, and the blood glucose in the empty abdomen was measured 2 times continuously to be 16.7mmol/L or more, and model of type 2 diabetic (T2 DM) mice was successful.
(2) Establishing obesity-related muscle-wasting mouse model
Male C57BL/6J mice (Shandong university model animal study center) were obtained at 6 weeks of age, and fed on a High Fat Diet (HFD) for 30 weeks from 8 weeks of age, to establish an obesity-related muscle-wasting-reduced mouse model.
For human umbilical cord mesenchymal stem cells (hucMSCs), human umbilical cord mesenchymal stem cells (1×10 6 cells/mouse) were suspended in PBS buffer and injected into both mouse models via tail vein every 7 days for 8 cycles.
For human umbilical cord mesenchymal stem cell-derived exosomes (MSC-EXO), human umbilical cord mesenchymal stem cell-derived exosomes were injected every 3 days into both mouse models by tail vein at 200 μg for 8 weeks.
(3) Establishing a model of disuse muscle-wasting mice
The left hind limb of a 6-week old male C57BL/6J mouse was fixed with a soft plastic coated steel wire for 2 weeks as follows: the steel wire tie with the soft plastic coating is cut into a length of 20-25 cm and is used for fixing the left hind limb of the mouse from the foot to the thigh, so that the knee joint is in an extending state, and the ankle joint is in a plantar flexion state, thereby establishing and obtaining the model of the disuse muscle-wasting mouse.
Then, human umbilical cord mesenchymal stem cells (1×10 in PBS buffer solution 6 cells/mouse) was injected into tibialis anterior and soleus muscles once every 7 days for a total of 4 cycles, resulting in an im+hucmscs treated group. The control group (im+pbs treated group) was then given the same volume of PBS buffer injected without human umbilical cord mesenchymal stem cells.
Method 1: metabolic testing and in vivo muscle performance analysis
The body composition of each mouse model was measured using a dual energy X-ray absorber. The muscle mass ratio represents the percentage of muscle mass composition to body composition.
Mice were subjected to an intraperitoneal glucose tolerance test (IPGTT) and an intraperitoneal insulin tolerance test (IPITT), and glucose and insulin resistance of the mice were measured.
The running machine is used for measuring the running distance, and the running distance is specifically: after two days of acclimation, the motor ability of the mice was determined by measuring their ability to run to fatigue, and the mice were first allowed to run at 8 m/min for 20min. Thereafter the speed of the treadmill was increased by 0.2m/min. Fatigue is defined as the hind limb contacting the electric grid for more than 10 seconds (electric grid) is the device that punishes for false responses in animal learning experiments and gives electric shock to animals.
The grip strength is measured by using an electronic grip dynamometer, specifically: the mice were trained to grasp the horizontal grid with the grip dynamometer attached to each limb and pull back gently in a horizontal direction parallel to the grip dynamometer, the force applied to the grid each time before the mice lost grip was recorded, 3 grip trials were performed per mouse, 3 measurements were recorded and the average was taken.
Method 2: histological and immunostaining
(1) After anesthesia of the mice, the bilateral Tibialis Anterior (TA) was dissected and weighed. One muscle was treated with liquid nitrogen and stored in a-80 ℃ refrigerator, the other was fixed in 4% paraformaldehyde and hematoxylin-eosin (H & E) staining and immunostaining were performed.
(2) A 4% paraformaldehyde fixed TA muscle sample was embedded in paraffin and sectioned at 5 μm thickness in maximum cross section. After dewaxing, the sections were H & E stained according to standard procedures.
(3) For immunohistochemical staining, antigen retrieval was performed using antigen retrieval liquid after section dewaxing. Endogenous peroxidase was inactivated with 3% hydrogen peroxide for 15min, and after blocking in blocking solution (10% goat serum) for 30min, anti-fast and slow muscle fiber antibodies were added dropwise and incubated overnight at 4 ℃. The following day sections were incubated with secondary antibody for 60min at room temperature and then stained with 3,3' -Diaminobenzidine (DAB) solution. Images were taken using a microscope and the cross-sectional area (CSA) of the muscle fibers and the ratio of fast to slow muscle fibers were calculated using Image-Pro Plus software.
(4) For immunofluorescent staining, antigen retrieval was performed using antigen retrieval solution after section dewaxing. After blocking in blocking solution (10% goat serum) at room temperature for 30min, LC3 antibody was added dropwise and incubated overnight at 4 ℃. The next day was incubated with goat anti-rabbit FITC secondary at room temperature for 60min, followed by staining with DAPI for 5min. Fluorescent images were observed and captured using a fluorescent microscope. The proportion of LC3 positive myofibers was analyzed and calculated using ImageJ software.
Method 3: exosome tracing
For ex vivo tracking, MSC-EXO was labeled with Cy7 and injected into mice via tail vein. Mice were imaged using an In Vivo Imaging System (IVIS) 12 hours after injection. For in vitro exosome tracing, MSC-EXO was labeled using PKH67 green fluorescent cell ligation kit, then incubated with fully differentiated myotubes for 24 hours, and fluorescent signals were detected and captured by fluorescence microscopy.
Method 4: cell lines and RNAi
Human Embryonic Lung Fibroblasts (HELFs) were obtained from the China center for cell culture (Shanghai, china) and were cultured at 37℃with 5% CO 2 In the incubator (C), the culture was performed using a high-sugar DMEM medium supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin.
Mouse C2C12 myoblasts were obtained from the chinese infrastructure cell resource pool and cultured using high sugar DMEM medium supplemented with 10% fetal bovine serum and antibiotics.
After the degree of fusion reached 80% -90%, the medium was replaced with a differentiation medium consisting of dmem+2% heat inactivated horse serum and antibiotics, with the medium being refreshed every 2 days for 4 days. Fully differentiated myotubes were stimulated with palmitic acid (0.6 mM) for 24 hours and then passed through a Transwell TM The system was co-cultured with or incubated with derived exosomes (25 μg/ml) of human umbilical cord mesenchymal stem cells for 24h.
To further explore the mechanisms of human umbilical mesenchymal stem cells and their derived exosomes, myotubes were pre-treated with small interfering RNAs (siRNA) or autophagy inhibitors 3-methyladenine (3-MA, 2 mM) prior to hucMSCs and MSC-EXO treatment.
siRNA oligonucleotides were synthesized by GenePharma, inc. (Shanghai, china). Negative Control (NC) siRNA sequences were:
sense5'-UUCUCCGAACGUGUCACGUTT-3'（SEQ ID NO.1）、
antisense 5'-ACGUGACACGUUCGGAGAATT-3'（SEQ ID NO.2）。
the AMPK alpha 2 siRNA sequence is:
sense 5'-CCCAGAUGAACGCUAAGAUTT-3'（SEQ ID NO.3）、
antisense5'-AUCUUAGCGUUCAUCUGGGTT-3'（SEQ ID NO.4）。
ampkα2 siRNA transfection was performed on C2C12 myotubes using Lipofectamine2000 transfection reagent. Briefly, C2C12 myoblasts were plated on 6-well plates and induced to fully differentiate, then the medium was removed and transfected with Opti-MEM I medium containing siRNA-AMPKα2 (125 nM) and Lipo2000 for 6 hours. Then replaced with differentiation medium and treated with hucMSCs or MSC-EXO. After 24h, myotubes were collected for subsequent experiments.
Method 5: immunoblot analysis
The mouse tibialis anterior muscle samples were lysed with RIPA lysate and protein concentration was measured by BCA method. Subsequently, the proteins were isolated and transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH 00010,0.45 μm microwells), the membranes were blocked with 5% skim milk for 1 hour at room temperature, and incubated overnight in a specific primary antibody at 4 ℃. After incubation of horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature the next day, protein bands were detected using chemiluminescence. The main antibodies are: atrogin1 (protein, china, cat No. 67172-1-Ig, 1:5000), muRF1 (protein, cat No. 55456-1-AP, 1:1000), AMP-activated protein kinase (AMPK, USA, CST, cat No. 5831S, 1:1000), AMP-activated protein kinase (Thr 172) (CST, cat No.2535S, 1:1000), ULK1 (Bioss, china, cat No. bs-3602R, 1:500), phospho- (p-) ULK1 (Immunoway, USA, cat No. YP1542, 1:1000), LC3 (protein, cat No. 14600-1-AP, 1:1000), p62 (Abcam, USA, cat No. 91526, 1:1000), GADPH (Boster, china, cat No. 3:500).
Method 6: RNA sequencing (RNA-seq) and real-time quantitative PCR analysis
Total RNA was isolated using Trizol reagent (Invitrogen, USA). Library construction and sequencing work was done by Shenzhen megagene Inc. STAR (v.2.5) was used to index the mouse reference genome (mm 10) and align the resulting fastq files. Differential expression analysis was performed using DESeq2 (v1.28.1). KEGG pathway enrichment was performed by cluster analyzer clusterifier (v3.10.1). 1. Mu.g of RNA was reverse transcribed using a reverse transcription kit (Cat. No. RR047A; takara, japan). Primers were chemically synthesized by Shanghai Ji Ma pharmaceutical technologies.
The primer sequences were as follows:
fbxo32: sense 5'-GGGGTCACCCTGCAGCTTTGC-3' (SEQ ID NO. 5) and anti 5'-GGGGAAAGTGAGACGGAGCAGC-3' (SEQ ID NO. 6);
trim63: sense5'-ATGGACCGGCACGGGGTGTA-3' (SEQ ID NO. 7) and anti 5'-GCACATCGGGTGGCTGCCTT-3' (SEQ ID NO. 8);
ulk1: sense 5'-AAGTTCGAGTTCTCTCGCAAG-3' (SEQ ID NO. 9) and anti 5'-CGATGTTTTCGTGCTTTAGTTCC-3' (SEQ ID NO. 10);
map1lc3b: sense 5'-TTATAGAGCGATACAAGGGGGAG-3' (SEQ ID NO. 11) and anti 5'-CGCCGTCTGATTATCTTGATGAG-3' (SEQ ID NO. 12);
sqstm1: sense 5'-AGGATGGGGACTTGGTTGC-3' (SEQ ID NO. 13) and anti 5'-TCACAGATCACATTGGGGTGC-3' (SEQ ID NO. 14);
gapdh: sense 5'-AAGGGCTCATGACCACAGTC-3' (SEQ ID NO. 15) and anti-sense 5'-CAGGGATGATGTTCTGGGCA-3' (SEQ ID NO. 16).
The real-time polymerase chain reaction was performed using SYBR Green PCR kit (Cat.No. RR420A; takara), the gene expression change was determined based on the relative quantification method comparing Ct values, and normalized quantification was performed by using Gapdh as a control.
Statistical analysis
All data are expressed as mean ± standard error. The differences between the two groups were tested statistically using either unpaired t-test or one-way anova, followed by GraphPad Prism 8 software. Differences were considered statistically significant at P < 0.05.
Analysis of results
Example 1: hucMSCs treatment group
hucMSCs were injected by tail vein every 7d (1×10) using mouse model 1 6 Individual/mouse, suspended in 200 μl PBS) to db/db mice at 5 weeks of age, 8 injections total.
Comparative example 1: PBS treatment group
The hucMSCs solution of example 1 was replaced with PBS buffer and injected into db/db mice at 5 weeks of age.
After 1 week of the 8 th injection of example 1 and comparative example 1, example 1 and comparative example 1 were tested using metabolic testing and in vivo muscle performance analysis methods (method 1) and histological and immunostaining (method 2), respectively, as follows:
(1) The isolated hucMSCs were identified by flow cytometry, adipogenesis and osteogenic induced differentiation, and the results are shown in FIG. 1, wherein the hucMSCs showed positive expression of CD105 and CD73 on the surface, negative expression of CD34 and HLA-DR, and the hucMSCs had adipogenic and osteogenic multipotential differentiation ability, and were in line with the characteristics of stem cells.
(2) Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed, and the results are shown in part A-B of FIG. 2, and hucMSCs improved glucose and insulin resistance.
(2) The grip strength was measured using an electronic grip dynamometer, and the results are shown in part C of FIG. 2, where the muscle strength of db/db mice was reduced compared to db/m mice, and hucMSCs injection was improved.
(3) The dual energy X-ray examination of the mouse muscle proportion, the isolated muscle weighing, and as shown in the D-F part of fig. 2, the injection of hucMSCs had no effect on body weight, but improved the muscle tissue proportion and the quality of Tibialis Anterior (TA).
(4) Histological H & E staining, immunostaining and molecular index detection were performed, resulting in G-L fractions as shown in fig. 2, db/db mice muscle fiber cross-sectional area (CSA), including fast and slow muscle fibers, significantly less than db/m mice; the hucMSCs can relieve the decrease of muscle fiber CSA caused by diabetes, increase the proportion of slow muscle fiber and promote the formation of muscle fiber; as shown in part M of FIG. 2, injection of hucMSCs also reduced the expression of E3-ubiquitin ligase Atrogin1 and MuRF 1.
The above results indicate that injection of hucMSCs can reduce diabetes-induced db/db mice muscle atrophy without affecting body weight. Thus, hucMSCs can alleviate muscle atrophy caused by diabetes.
Example 2: HFD+hucMSCs treatment group
With the use of the mouse model 2,high fat diet feeding was started at 8 weeks of age, and hucMSCs (1×10) were injected by tail vein at 38 weeks of age 6 Individual/mouse, suspended in 200 μl PBS) to High Fat Diet (HFD) fed mice, were injected every 7 days for a total of 8 injections.
Comparative example 2: HFD+PBS treatment group
The hucMSCs solution in example 2 was replaced with PBS and 200 μl of PBS was injected into High Fat Diet (HFD) fed mice at 38 weeks of age by tail vein every 7 days for a total of 8 injections.
After 1 week from the last administration of example 2 and comparative example 2, example 2 and comparative example 2 were tested using metabolic testing and in vivo muscle performance analysis methods (method 1) and histological and immunostaining (method 2), respectively, as follows:
(1) Exercise tolerance and grip strength were measured by intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) using a treadmill and an electronic grip meter, and as a result, hucMSCs improved glucose and insulin tolerance and enhanced exercise tolerance and grip strength as shown in parts a-D of fig. 3.
(2) The results were measured by histological and immunostaining methods as follows:
as in parts E-F of fig. 3, hucMSCs reduced weight gain induced by high fat diets and increased muscle mass fraction; as in part G of fig. 3, injection of hucMSCs can increase the quality of the mouse tibialis anterior.
Meanwhile, as shown in the H-M part of fig. 3, injection of hucMSCs can also increase the cross-sectional area of muscle fibers, particularly fast muscle fibers, and can increase the percentage of slow muscle fibers and the number of muscle fibers; as in part N of FIG. 3, injection of hucMSCs inhibited the upregulation of muscle atrophy-associated Atrogin1 and MuRF1 protein levels.
The above results indicate that injection of hucMSCs can reduce obesity-induced muscle atrophy.
Example 3: im+hucmscs treatment group
hucMSCs (1×10) were injected locally into the braked limb of mouse model 3 6 Mice/mouse suspended in 200 μl PBS), 1 injection every 7 days for 4 times.
Comparative example 3: im+pbs treatment group
The hucMSCs solution in example 3 was replaced with PBS and the braked limb was locally injected with 200 μl of PBS 1 every 7 days for a total of 4 times.
Example 3 and comparative example 3 were tested using metabolic testing and in vivo muscle performance analysis methods (method 1) and histological and immunostaining (method 2), respectively, as follows:
(1) The grip strength of the mice was measured using a treadmill and an electronic grip dynamometer, and as shown in section a-B of fig. 4, the exercise endurance and grip strength of the mice injected with hucMSCs of example 3 were improved compared to comparative example 3.
(2) The results were measured by histological and immunostaining methods as follows:
as in the C-D section of FIG. 4, hucMSCs were able to increase the ratio of the muscle mass and the mass of the tibialis anterior muscle in mice.
As in the E-K portion of FIG. 4, hucMSCs also increased the cross-sectional area of mouse muscle fibers, including the fast and slow muscle fibers of IM mice, increased the percentage of slow muscle fibers and the number of muscle fibers, and reduced brake-induced upregulation of Atrogin1 and MuRF1 expression.
The above results indicate that hucMSCs can ameliorate muscle atrophy induced by braked limbs (disuse muscle atrophy), further indicating that the anti-muscular atrophy effect of hucMSCs may be due to its direct effect on muscles, rather than an indirect effect by improving body metabolism.
Example 4: db/db+hucmscs treated group, hfd+hucmscs treated group, im+hucmscs treated group
The injection of hucMSCs (1×10) was performed on mouse model 1, mouse model 2 and mouse model 3, respectively 6 Individual/mouse, suspended in 200 μl PBS).
Comparative example 4: db/db+pbs treated group, hfd+pbs treated group, im+pbs treated group
The hucMSCs solution in example 4 was replaced with PBS, and mice model 1, 2 and 3 were treated with 200 μl PBS, respectively.
The tibialis anterior muscle of model 1 mice injected with PBS buffer or hucMSCs in example 4 and comparative example 4, respectively, was tested using high throughput sequencing technology (RNA-seq) and real-time quantitative PCR analysis (method 6), as follows:
(1) High throughput sequencing showed 1426 Differentially Expressed Genes (DEGs), as shown in fig. 5 a, with 627 genes up-regulated and 799 genes down-regulated in model 1 mice injected with hucMSCs relative to control injected with PBS buffer; KEGG pathway analysis showed that DEGs was enriched in AMPK and autophagy signaling pathways as shown in B of fig. 5.
(2) Model 1 mice were analyzed by RT-qPCR and as shown in section C-D of fig. 5, injection of hucMSCs was able to down-regulate muscle wasting-related genes Fbxo32 (Atrogin 1), trim63 (MuRF 1) and autophagy-related genes Ulk1, map1lc3b and Sqstm1.
(3) In db/db mice, the levels of AMP-activated protein kinase (AMPK) (T172) and downstream ULK1 (S555) phosphorylation were lower than in db/m mice, as shown in part A of FIG. 6, and the levels of phosphorylation could be up-regulated after hucMSCs injection, while the ratio of LC3-II to LC3-I and p62 content were significantly higher in db/db mice than in db/m mice, but could be significantly down-regulated in hucMSCs dry prognosis.
Furthermore, immunofluorescent staining of LC3 showed that injection of hucMSCs reduced the proportion of LC3 positive myofibers, indicating that hucMSCs increased autophagy levels of skeletal muscle and restored autophagy flux, as shown in part B of fig. 6.
(4) Analysis of model 2 high fat diet mice and model 3 brake mice demonstrated that hucMSCs intervention also promotes phosphorylation of AMPK (T172) and ULK1 (S555) and reduces LC3-II to LC3-I ratio, p62 content and LC3 positive myofibers ratio as in the C-F section of fig. 6.
The above results indicate that hucMSCs are capable of activating the AMPK/ULK1 signaling pathway, promoting autophagy of skeletal muscle.
Example 5: pa+hucmscs treatment group
Myotube model induced in vitro with C2C12, treated with 0.6mM Palmitate (PA) induced myotube atrophy. PA can mimic lipotoxicity and induce myotube atrophy.
Example 5 was tested using autophagy inhibitor 3-MA or AMPK targeted siRNA (method 4), immunoblotting (method 5), and specifically as follows:
(1) The results are shown in fig. 7a by immunoblot analysis:
at this dose, PA treatment significantly promoted the expression of Atrogin1 and MuRF1, while hucMSCs treatment was able to eliminate PA-induced increases in Atrogin1 and MuRF1 expression, while PA reduced the phosphorylation levels of AMPK (T172) and ULK1 (S555), increasing LC3-II to LC3-I ratio and p62 content in myotubes. The use of hucMSCs treatment was able to significantly rescue PA-induced AMPK/ULK1 signaling of C2C12 myotubes, reducing the content of LC3II/LC3I and p62, indicating an autophagy process and an increase in autophagy flux.
Meanwhile, as shown in fig. 7B, the hucMSCs treatment can also increase the diameter of myotubes.
Thus supporting the direct cellular autonomy of hucMSCs on myotubes.
(2) Transfection assays were performed using targeted siRNA for autophagy inhibitor 3-MA or AMPK, and immunoblot analysis results are shown in figure 7, section C, with hucMSCs-mediated upregulation of ULK1 phosphorylation and reduction of LC3II/LC3I and p62 levels in the presence of 3-MA being partially eliminated; as shown in part D of FIG. 7, 3-MA was able to reduce the hucMSCs-mediated decline in the levels of Atrogin1 and MuRF1 and increase in myotube diameter.
(3) As shown in E-F of FIG. 7, after silencing AMPK by siRNA, the effect of hucMSCs on improvement of p-AMPK/p-ULK1, LC3/p62, atrogin1/MuRF1 and myotube diameter was reduced.
The above results indicate that hucMSCs alleviate PA-induced muscle atrophy by promoting AMPK-mediated autophagy in the C2C12 myotubes.
Example 6: MSC-EXO treatment group
MSC-EXO (200. Mu.g in 200. Mu.l PBS) was injected into db/db mice of mouse model 1 by tail vein every 3d for 8 weeks.
Comparative example 6: PBS treatment group
The hucMSCs solution in example 6 was replaced with PBS and 200 μl PBS was injected into db/db mice of mouse model 1 every 3d for 8 weeks.
Example 6 was tested using metabolic testing and in vivo muscle performance analysis methods (method 1) and histological and immunostaining (method 2), exosome tracking (method 3), immunoblotting (method 5), as follows:
(1) As shown in A-B of FIG. 8, MSC-EXO with a diameter of about 130nm isolated from hucMSCs culture supernatant was in a circular bilayer membrane structure with complete coating by nanoparticle size analysis and Transmission Electron Microscope (TEM) analysis.
Immunoblotting results are shown in FIG. 8C, CD9, CD63 and CD81 were enriched in MSC-EXO, while endoplasmic reticulum marker Calnexin was enriched in hucMSCs and deleted in MSC-EXO.
(2) The distribution of MSC-EXO in vivo was detected after 12h by performing exosome tracking using Cy 7-labeled MSC-EXO and injecting into mice. The results are shown in FIGS. 8D-E, where mice were imaged by an In Vivo Imaging System (IVIS), which images show that MSC-EXO can reach skeletal muscle.
(3) The grip strength was measured by intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) using an electronic grip dynamometer, and as a result, MSC-EXO injection improved glucose and insulin resistance and grip strength as shown in A-C of FIG. 9.
(4) As measured by histological and immunostaining methods, as shown in D-F of fig. 9, MSC-EXO intervention had no effect on body weight, but increased the muscle mass fraction and increased the mass of tibialis anterior; as shown in G-L of FIG. 9, MSC-EXO also increases the cross-sectional area (CSA) of db/db mouse muscle fibers, including fast and slow muscle fibers, increasing the percentage of slow muscle fibers and the number of muscle fibers.
(5) As measured by immunoblotting, MSC-EXO was able to promote phosphorylation of AMPK (T172) and ULK1 (S555) while reducing the content of LC3II/LC3I and p62 and the proportion of LC3 positive myofibers, as shown in M-N of FIG. 9.
The above results indicate that injection of MSC-EXO can prevent diabetes-induced muscle atrophy and enhance the AMPK/autophagy signaling pathway, consistent with the effects of hucMSCs.
Example 7: PA+MSC-EXO treatment group
Fully differentiated C2C12 myotubes were treated with 25 μg/ml MSC-EXO for 24 hours in the presence and absence of targeted siRNA to autophagy inhibitor 3-MA or AMPK.
Example 7 was tested using exosome tracking (method 3), cell lines and RNAi (method 4), immunoblotting (method 5) respectively, as follows:
(1) For in vitro exosome tracing, MSC-EXO was labeled using PKH67, and fluorescent signals were detected and captured by fluorescence microscopy, as shown in fig. 10 a, and the labeled exosomes were found to be able to be taken up by the C2C12 myotubes.
(2) By immunoblotting analysis, as shown in FIG. 10B, MSC-EXO treatment was able to reduce PA-induced expression of Atrogin1 and MuRF1, and at the same time, was able to promote phosphorylation of AMPK (T172) and ULK1 (S555), and reduce the content of LC3II/LC3I and p 62.
(3) As shown in FIG. 10C, treatment of fully differentiated C2C12 myotubes with MSC-EXO can increase the diameter of the myotubes.
(4) By immunoblot analysis, the results are shown in FIG. 10D, where 3-MA is capable of partially eliminating MSC-EXO mediated upregulation of ULK1 phosphorylation levels and reduction of LC3II/LC3I and p62 levels; as shown in D-E of FIG. 10, 3-MA was able to reduce MSC-EXO mediated decreases in the levels of Atrogin1 and MuRF1 and increases in myotube diameter.
(5) As shown in F-G of FIG. 10, silencing AMPK with siRNA attenuated the effect of MSC-EXO on p-AMPK/p-ULK1, LC3/p62, atrogin1/MuRF1 and myotube diameter.
The above results indicate that MSC-EXO can alleviate muscle atrophy by promoting AMPK-mediated autophagy.
The foregoing description of the preferred embodiments of the present disclosure is provided only and not intended to limit the disclosure so that various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
While the disclosure has been described in detail in connection with the drawings and embodiments, it should be understood that the foregoing description is not intended to limit the disclosure in any way. Modifications and variations of the present disclosure may be made as desired by those skilled in the art without departing from the true spirit and scope of the disclosure, and such modifications and variations fall within the scope of the disclosure.
Claims (10)
1. Use of an exosome in the manufacture of a medicament for treating muscle atrophy induced by type 2 diabetes, wherein the exosome is umbilical cord mesenchymal stem cell-derived exosome that ameliorates muscle atrophy induced by type 2 diabetes by enhancing AMPK/ULK 1-mediated autophagy.
2. The use according to claim 1, wherein the method of preparation of the exosomes comprises:
obtaining human umbilical cord tissue;
culturing human umbilical cord tissue by using an alpha-MEM culture medium until the cell reaches 80% of fusion;
culturing in exosome-free culture medium, and collecting to obtain supernatant;
and centrifuging the supernatant to obtain the exosome.
3. The use according to claim 2, wherein the alpha-MEM medium contains 10% fetal bovine serum, 100U/mL penicillin and 100 μg/mL streptomycin.
4. The use according to claim 2, characterized in that the supernatant is filtered through a 0.22 μm filter and centrifuged.
5. The use according to claim 4, wherein the supernatant is filtered through a 0.22 μm filter and centrifuged at 120000g for 70 minutes to obtain the exosomes.
6. The use according to claim 1, wherein the exosomes increase the cross-sectional area and number of muscle fibers to improve muscle atrophy caused by type 2 diabetes.
7. The use of claim 6, wherein the exosomes modulate the ratio of fast and slow muscle fibers to ameliorate muscle atrophy caused by type 2 diabetes.
8. The use of claim 1, wherein the exosomes promote phosphorylation of AMPK and ULK1 to enhance AMPK/ULK1 mediated autophagy.
9. Use of an exosome, which is umbilical cord mesenchymal stem cell-derived exosome, for the preparation of a medicament for treating muscle atrophy caused by obesity, the exosome improving muscle atrophy caused by obesity by enhancing AMPK/ULK 1-mediated autophagy.
10. Use of an exosome in the manufacture of a medicament for treating disuse muscle atrophy, wherein the exosome is umbilical cord mesenchymal stem cell-derived exosome that ameliorates disuse muscle atrophy by enhancing AMPK/ULK 1-mediated autophagy.
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