CN113069470A - Application of brown adipocyte-derived exosome - Google Patents

Abstract

The invention discloses application of exosomes derived from brown adipocytes. Application of brown adipocyte-derived exosome in preparation of medicine for treating non-alcoholic fatty liver disease, preferably application in preparation of medicine for treating non-alcoholic fatty liver disease caused by high fat obesity. The brown adipocyte-derived exosomes surface reach the marker proteins CD63, CD9 and TSG 101. The extracted brown fat cell-derived exosome can effectively reduce the weight of the non-alcoholic fatty liver of a high-fat-induced obese mouse, the ratio of the liver to the volume, the content of fatty acid and cholesterol in the liver, the liver is reduced, the synthesis of fat in the liver is slowed down, and the consumption of fatty acid is increased. The method provides a new way for treating the nonalcoholic fatty liver disease of fat mice caused by high fat.

Description

Application of brown adipocyte-derived exosome

Technical Field

The invention relates to application of exosomes derived from brown adipocytes, in particular to application of exosomes generated by brown adipocytes in treatment of non-alcoholic fatty liver caused by obesity.

Background

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease defined as the accumulation of fat in more than 5% of the hepatocytes of the liver without or with small amounts of alcohol. According to incomplete statistics, 14% to 27% of people worldwide have fatty liver. The current drug treatment for fatty liver does not meet the clinical requirements, and to date, there is no FDA approved drug for the treatment of more severe non-alcoholic fatty liver disease.

Brown adipose tissue is a tissue that generates energy, which can pass through the uncoupling protein UCP1 in mitochondria, thereby converting chemical energy stored in the body into thermal energy to maintain body temperature, i.e., generate non-tremor heat to maintain body temperature. With the concomitant re-discovery of brown adipose tissue in adults, studies have shown that the presence of brown fat in humans is inversely related to obesity and diabetes, and thus the activation of brown adipose tissue has received increasing attention as a means of treating metabolic diseases. And recent studies have shown that transplantation of brown adipose tissue can not only improve insulin resistance in high fat-induced obese mice, but also reduce glucose homeostasis in naturally obese mice ob/ob (leptin-deficient mice) without affecting feeding. However, since brown adipose tissue exists in a mixed state with white adipose tissue in a human body, which is mainly present in the vicinity of scapula, perirenal and spinal columns, and has no distinct and distinguishable tissue masses as in rodents, and the survival rate of transplanted adipose tissue is low, the treatment of fatty liver and other metabolic diseases by transplantation of brown adipose tissue has great technical difficulty in a human body. At the same time, the treatment by means of stimulating the human body's brown adipose tissue by cold or other stimulation means to produce heat and consume energy has a great limitation, because some obese human bodies have no or little brown fat, so that the energy consuming capacity is limited. Recent studies have shown that brown adipose tissue is an important endocrine organ in addition to a heat-producing organ, and can secrete a variety of brown fat factors to regulate the metabolic homeostasis of the whole body. Among them, exosomes (exosomes) are gaining increasing attention as an important intercellular communication tool. The exosome carries a large amount of nucleic acid (miRNA, lncRNA, mRNA), protein and lipid to reach a target cell for regulation, and meanwhile, the diameter of the exosome accords with the size of a nanometer material, so that the exosome has strong capability of delivering information, and meanwhile, the exosome is endogenous and cannot cause immune reaction of a body. However, reports of treating fatty liver by brown adipocyte-derived exosomes are not seen at present.

Disclosure of Invention

The invention aims to provide application of brown adipocyte-derived exosomes in preparation of a medicament for treating non-alcoholic fatty liver disease.

The purpose of the invention can be realized by the following technical scheme:

application of brown fat cell-derived exosome in preparation of medicine for treating non-alcoholic fatty liver disease.

Preferably, the invention relates to application of exosome derived from brown fat cells in preparing a medicament for treating non-alcoholic fatty liver disease caused by high fat obesity.

The brown adipocyte-derived exosomes surface reach the marker proteins CD63, CD9 and TSG 101. The results of particle lens and electron microscope show that the average diameter of the particle lens is about 100nm, and the results of electron microscope show that the particle lens has the characteristic that the lipid bilayer structure conforms to exosome.

The invention is proved by animal experiments for the first time that: the exosome secreted by the brown adipocyte can effectively improve the nonalcoholic fatty liver of fat mice caused by high fat, and comprises the following components of reducing the weight of the liver, the ratio of the liver to the body weight, reducing the content of lipid droplets in a liver slice, reducing the size of the lipid droplets, reducing the content of triglyceride and total cholesterol in the liver, simultaneously reducing the expression level of genes related to fatty acid synthesis in the liver and increasing the expression level of genes related to fatty acid beta oxidation.

The invention has the beneficial effects that:

1. the extracted brown fat cell-derived exosome can effectively reduce the weight of the non-alcoholic fatty liver of a high-fat-induced obese mouse, the ratio of the liver to the volume, the content of fatty acid and cholesterol in the liver, the liver is reduced, the synthesis of fat in the liver is slowed down, and the consumption of fatty acid is increased. The method provides a new way for treating the nonalcoholic fatty liver disease of fat mice caused by high fat.

2. Exosomes act as endogenous extracellular vesicles with diameters similar to nanoscale vectors. And exosomes are capable of carrying different signaling molecules (RNA and protein), and thus have potential as drug delivery vehicles. Compared with exogenous nano-carriers, the exosome has the advantages of no immune response, no biological toxicity and the like. The exosome extracted by the method can be stored in a refrigerator at minus 80 ℃ for a long time.

Description of the drawings:

FIG. 1 is a Western-blot method for detecting marker proteins CD63, CD9 and TSG101 of brown adipocyte-derived exosomes.

Figure 2 electron microscopy observation of exosome morphology: the scale is 100 nm.

FIG. 3 particle lens detects the diameter distribution of exosomes.

FIG. 4 schematic diagram of a plan for exosome treatment of non-alcoholic fatty liver disease.

Figure 5 mouse body weight change before and after treatment.

FIG. 6 weight of liver and ratio of liver weight to body weight in treated and non-treated groups. P < 0.05; p < 0.01; p <0.001

Figure 7 pathological changes in liver of treated and non-treated high fat mice: h & E stained section with 100 μm scale.

Figure 8 content of lipid droplets in liver of treated and non-treated high fat mice: oil red staining pattern, scale 100 μm.

FIG. 9 variation of triglyceride content in liver of high-fat mice in treated group and non-treated group. P < 0.05; p < 0.01; p <0.001

FIG. 10 change in total cholesterol content in liver of high-fat mice in treated group and non-treated group. P < 0.05; p < 0.01; p <0.001

FIG. 11 shows the changes in the expression levels of genes involved in cholesterol synthesis, efflux, fatty acid absorption, synthesis and β oxidation in the liver of mice with high fat in the treated group and in the non-treated group. P < 0.05; p < 0.01; p <0.001

The specific embodiment is as follows:

example 1 exosomes secreted by cultured brown adipocytes were isolated.

(1) Extraction of brown adipocyte exosomes

After the mice were sacrificed by neck-breaking, brown adipocytes were taken out from the scapular region of the mice, and a small amount of white fat above the brown fat was removed. The brown adipocytes were minced with scissors. After cutting, the cells were placed in a culture medium of DMEM (all available from Gibco) containing 10% mv free FBS, and then placed in a carbon dioxide incubator for culturing. After 24 hours of culture, the culture broth was collected and subjected to extraction of exosomes.

(2) Exosome extraction

Firstly, pretreating the culture solution by 300Xg, centrifuging for 10 minutes, removing 3000Xg after precipitation, centrifuging for 30 minutes, removing sediment, finally 10000Xg, centrifuging for 60 minutes, and removing the precipitate. The pretreatment is to remove cell debris or some larger vesicles such as apoptotic bodies, MV, etc. from the culture. Finally, the pretreated culture solution was subjected to ultracentrifugation at 120000Xg for 3 hours. After centrifugation, the culture was slowly poured off, and the exosomes at the bottom of the tube were then resuspended in 60ul of PBS for subsequent experiments.

Example 2 identification of Brown adipocyte-derived exosomes

(1) Western-blot identification of exosome marker proteins CD63, CD9 and TSG101

An appropriate amount of cell lysate was added. The cell lysate (purchased from Biyun day) was lysed on ice for 30 minutes to allow the cell lysate to act sufficiently. And carrying out ultrasonic disruption on cells in the cell lysate to enable the protein in the cells to be released more completely. Centrifuge at 12000Xg for 5 minutes at 4 ℃. The supernatant was aspirated and tested for protein concentration. The protein concentration was measured by BCA method (available from Saimer fly) by diluting the protein 10-fold, i.e.adding 1. mu.l of protein solution to 9. mu. lddH2O (see the relevant instructions for details). The protein was adjusted to the same concentration, 5x loading buffer (purchased from Byun), then boiled in a metal bath at 100 ℃ for 5 minutes, and then the boiled protein was stored at-20 ℃. SDS-PAGE gel (related reagents are purchased from Biyun day) is prepared, after protein samples are slowly loaded, electrophoresis is carried out by using a constant voltage of 70V in a gel concentration stage, and after the protein enters separation gel, the voltage is increased to 110V for constant voltage running. The time for running the gel is determined according to the molecular weight of the target protein. And (5) transferring the film. The PVDF membrane was first activated with methanol for one minute. Then the membrane is rotated according to the sequence of sponge-filter paper-glue-PVDF membrane-filter paper-sponge. The generation of bubbles should be avoided as much as possible during the film transfer process. The membrane transfer time is 90 minutes with a constant current of 300 mA. And (5) sealing after the film transfer is finished. 5% skim milk powder in TBST formulation was used as a block. Blocking at room temperature for 1 hour. After blocking, the strips were coated with primary antibody (CD63, CD9 and TSG101 from Santa Cruz) and placed in a refrigerator at 4 ℃ overnight. The primary antibody was recovered and then eluted with TBST under the conditions of 15 minutes for 4 washes with a single change. After washing the membrane, the strips were incubated with the corresponding secondary antibody at room temperature for 1 hour. Recovering the secondary antibody and washing the membrane. Washed once with TBST15 min and 4 times. And (6) exposing. The band was slightly blotted with a water-absorbent paper to remove the residual liquid, and then a luminescent substrate (purchased from semer plane) was added thereto, followed by exposure with an exposure machine, and the result was shown in fig. 1.

(2) Exosome morphology observed by electron microscope

The exosomes obtained by gradient centrifugation were resuspended in 4% paraformaldehyde, and then sent to the university of Nanjing medical sciences for electron microscopy, and the results are shown in FIG. 2, and the microparticles obtained by gradient centrifugation had a lipid bilayer structure.

(3) Particle lens analysis of exosome diameter distribution

The exosome is fully mixed, gradient dilution is carried out, the diluted volume is at least 1ml, then the exosome is slowly injected into a particle lens analyzer for detection, at least three times of tests are carried out, and the result is shown in figure 3, and the detected diameter is about 130.

The results of this example show that brown adipocyte-derived exosomes can be successfully obtained by gradient centrifugation, the exosomes have surface expression levels of the target proteins CD63, CD9 and TSG101, the average diameter is about 100, and the morphology observed by electron microscopy conforms to the characteristics of exosomes. Example 3 therapeutic effect of exosomes secreted by brown adipocytes on non-alcoholic fatty liver disease in high fat-induced obese mice C57/B6 mice used for 6 weeks, purchased from the university of Nanjing institute of model animals.

(1) Mouse model of high fat induced obesity

Male C57BL/6J mice 6 weeks old were fed a high fat diet with 60% fat content. The amount of the high-fat feed is about 2 days of food for the mice, and the high-fat feed needs to be replaced or supplemented in time because the high-fat feed is easy to oxidize and deteriorate. And the mice were placed at 25 ℃ room temperature, given enough water and kept on a 12-hour light 12-hour dark work. The high-fat molding time is 3 months.

(2) Treatment of alcoholic fatty liver in hyperlipidemic mice with exosomes secreted by brown adipocytes

As shown in fig. 4, we divided mice 3 months after high fat molding into two groups, treated group was injected with exosome secreted by brown adipocytes at tail vein, and control group was injected with PBS, and the number of mice per group was 8. When the mice were injected with exosome tail vein, the amount of exosome injected was 90 μ g per mouse per day for 7 consecutive days.

(3) Mouse weight and liver weight detection

The body weights of the mice before and after the treatment were measured, and as a result, as shown in fig. 5, the body weights of the mice in the treatment group and the control group were not changed and were maintained at about 42 g. By the time the mice die, we also measured the weight of the mouse liver, which is shown in fig. 6, and the weight of the liver of the treated mice is approximately 1.6g, which is significantly lower than 2g of the control mice. Meanwhile, the results of the ratio of the liver weight to the body weight show that the ratio of the liver weight to the body weight of the treated mice is about 38mg/g, while the ratio of the liver weight to the body weight of the control mice is about 42mg/g, as shown in fig. 6, the treated mice have significant differences compared with the control mice. (P < 0.05;. P < 0.01;. P <0.001)

(4) Histopathological examination of mouse liver

Morphological observations of H & E staining for liver tissue: the livers were fixed in 4% paraformaldehyde for at least 24 hours and then paraffin embedded according to conventional paraffin embedding procedures. The embedded paraffin blocks were first sliced to a thickness of 5 μm. The slices were then routinely dewaxed to water, i.e.: xylene for 10 minutes (twice, 10 minutes each); absolute ethyl alcohol for 3 minutes; 95% ethanol for 3 minutes; 90% ethanol for 3 minutes; 80% ethanol for 3 minutes; 70% ethanol for 3 minutes; 60% ethanol for 3 minutes; 50% ethanol for 3 minutes; 30% ethanol for 3 minutes; distilled water for 3 minutes. Hematoxylin stain for 1 minute, used to stain nuclei. The mixture was rinsed with tap water for 15 minutes. Red stain for 15 min, used to stain cytoplasm. Then dehydrating with conventional ethanol, removing xylene, air drying, and sealing with neutral gum. As a result, as shown in FIG. 7, the liver lipid droplet content of the treated mice was significantly decreased and the lipid droplet size was also decreased as compared with the control group.

Oil red staining the content of lipid droplets in the liver was observed: the liver was fixed with 4% paraformaldehyde for 24 hours. The liver was embedded with OCT and then cryosectioned to a thickness of 10 μm. The sections were air dried at room temperature and soaked in PBS for 10 min. The slices were placed in 70% ethanol for 2 minutes. Oil red stain for 15 minutes. (oil red stock solution at 4 ℃ is mixed with double distilled water in a ratio of 3: 2, and after uniform mixing, filtration is carried out to remove undissolved particulate matters). The sections were washed in 75% ethanol for 5 minutes. Wash with PBS for 5 min. After soaking in PBS, the sample was taken out and observed rapidly. As shown in FIG. 8, the liver lipid droplet content of the treated mice was significantly decreased compared to the control group.

(5) Content of triglyceride and Total Cholesterol in mouse liver

The content of triglyceride and total cholesterol in the liver of the mice is measured by using the kit for measuring triglyceride and total cholesterol purchased from Biyunyan, and the measurement results are shown in figures 9 and 10, the content of triglyceride in the liver of the mice in a treatment group is about 53mg/g liver, the content of total cholesterol is about 17mg/g liver, and the contents are obviously reduced compared with the control group. (P < 0.05;. P < 0.01;. P <0.001)

(6) Changes in the expression levels of genes involved in cholesterol synthesis, efflux, fatty acid absorption, synthesis and beta-oxidation in the liver

RNA of liver tissues is extracted, after reverse transcription, fluorescence quantitative PCR (polymerase chain reaction) is carried out, and the change of expression quantity of sterol synthesis (HMGCR, SREBP-1c), outflow (CYP7A, ABCG1), fatty acid absorption (FATP1, FABP1, CD36), synthesis (FAS, SCD1, ACC alpha, PPARy) and beta oxidation (PDK4, PPAR alpha, CPT-1 alpha, ACOX-1, LCAD, MCAD, UCP2) related genes is detected. As shown in fig. 11, the expression level of the gene involved in fatty acid synthesis and fatty acid synthesis was significantly decreased in the liver of the treated mice, while the expression level of the gene involved in fatty acid β oxidation was increased by cholesterol efflux, compared to the control group. (P < 0.05;. P < 0.01;. P <0.001)

The results of the above experimental examples illustrate that: the exosome from the brown fat cell can effectively improve the non-alcoholic fatty liver of an obese mouse caused by high fat, so the exosome has considerable application value in preparing a preparation for treating the non-alcoholic fat.

Statistical analysis

Statistical data are given as mean ± sd, statistically analyzed and plotted using GraphPad Prism 5 software (san diego, usa), and two comparisons were made using t-test, P <0.05 (.: there is a statistical difference; p <0.01 (. sup.) was significantly different; p <0.001(×) had very significant differences.

Claims (3)

1. Application of brown fat cell-derived exosome in preparation of medicine for treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized by the use of brown adipocyte-derived exosomes for the preparation of a medicament for treating non-alcoholic fatty liver disease caused by high-fat obesity.

3. The use according to claim 1, characterized in that the brown adipocyte-derived exosomes surface-reach the marker proteins CD63, CD9 and TSG 101.

Images