Introduction

The worldwide meat industry is experiencing rapid growth, particularly in the case of red meat like pork1. In recent years, the excessive focus on achieving higher growth performance and increasing the proportion of lean meat has led to a decline in the quality and nutritional value of pork, despite it being a significant protein source2. The development of white adipose tissue (WAT) and skeletal muscle (SkM) is crucial for determining the quality of pork3. The development of muscle cells determines muscle yield as well as muscle fiber type, while the development of adipocytes determines the deposition of adipose tissue, and together muscle and fat affect the quality and flavor of pork4,5,6,7. Excessive fat in pigs is undesirable as it significantly impacts the economic returns of contemporary pig breeding, given that meat quality is the primary factor influencing consumer preference for meat consumption8,9. The appropriate proportion of fat helps to enhance the taste and flavor of pork, while also aiding in the absorption and utilization of nutrients by the human body. However, excessive fat content may increase the risk of obesity and cardiovascular disease. Insufficient intramuscular fat and excessive subcutaneous fat are top pork quality challenges10.

Obesity and exercise have a significant impact on WAT and SkM, making them the primary subjects of tissue-level studies11,12,13. Pedersen, proposed the idea that SkM acts as an immunogenic generator/secretor of cytokines, which have an endocrine role and should be categorized as myokines14. The mechanism of interdependent and closely linked dialogue between muscle and fat, on the one hand, WAT serves as an important fuel reservoir for the animal organism, on the other hand, providing energy for other tissues such as SkM. Conversely, exercises targeting SkM have an impact on the endocrine system, influencing adipocytes through various factors such as irisin, myostatin, β-aminoisobutyric acid, fibroblast growth factor 21 (FGF-21), and lactate15. The function of WAT is regulated by all the aforementioned muscle factors.

The SkM acts as an endocrine organ, producing and releasing a range of myokines/sEV to control metabolism in the SkM and surrounding tissues through autocrine/paracrine/endocrine mechanisms16,17,18. Systemic messengers known as extracellular vesicles (EV) play a crucial role in transmitting signaling molecules that facilitate communication between cells and organs19. Extracellular vesicles (EVs), including exosomes and microvesicles, which are produced through various biogenesis pathways, are considered to be crucial20,21. Cells generate extracellular vesicles, which are nanoparticles22. Comprising the cellular membrane and encompassing membrane proteins, they enclose a watery center comprising soluble molecules like proteins and nucleic acids, including miRNA and mRNA23. These processes play a crucial role in numerous physiological and pathological events by facilitating the transfer of biological molecules from cells that produce them to cells that receive them24.

The interaction between muscle and fat is complex, but instead of and there is no existing report on the involvement of sEV in regulating the association between muscle and fat in pigs. Hence, this study aims to investigate the connection and fusion between the two tissues of pig muscle and fat using sEV as a mediator. The main focus will be identifying and confirming the function of miRNAs transported by sEV derived from muscle and fat. Functional restoration through oleic acid and insulin confers complex heterogeneity of sEV, with miR-146a-5p as the key miRNA. The findings of this research will be precious in uncovering the network mechanism behind the development of pork quality characteristics.

Materials and methods

Animals and sample collection

From the Guangzhou Purebred Pig Farm in Guangzhou City, Guangdong Province, China, four male Landrace pigs that were three days old and in good health were chosen. The pigs were anesthetized via electrical stunning and sacrificed by exsanguination. The longissimus dorsi (LD) muscle and subcutaneous adipose tissue (SAT) were dissected and taken to the lab to be transferred to a DMEM-F12 medium (Gibco, New York, NY, USA).

The isolation and culture of porcine MuSCs

Small sections of porcine muscle samples were cut and then moved into DMEM-F12. The trimmed tissues were digested with 0.2% collagenase type II for one hour. Subsequently, the treated tissue was centrifuged at 1500 g for 10 min at a temperature of 4 °C. The precipitate was resuspended in DMEM-F12. The sediment was reconstituted in DMEM-F12 and underwent three rounds of centrifugation at 800 g, 4 °C, for 10 min. Afterward, the suspension of cells was passed through a cell strainer with a pore size of 75 μm. After filtration, the supernatant was subjected to centrifugation at a speed of 800 g and a temperature of 4 °C for 5 min. The lower pellet was suspended in DMEM-F12 solution and placed in a cell culture flask for one hour at a temperature of 37  °C and a CO2 concentration of 5%. Skeletal muscle progenitor cells stay suspended in the supernatant, whereas fibroblasts rapidly attach to the base of the cell culture flask. In the end, porcine MuSCs were cultivated in DMEM-F12 (20% FBS) at a temperature of 37 degrees Celsius with a 5% concentration of CO2.

Isolation and culturing of porcine primary adipocyte

The subcutaneous fat tissue was divided into sections measuring 1 mm3 and then moved to the DMEM-F12 medium. The tissue sample was treated with 0.2% collagenase type II (Gibco, New York, NY, USA) for 2 h at a temperature of 37 °C while being agitated. After passing through a 150 μm mesh, the digested tissue was filtered, and the resulting liquid was then centrifuged at 600 g for 10 min. The pellet was mixed with Erythrocyte Lysis Buffer (Sangon Biotech, Shanghai, China) and left undisturbed for 10 min to break down the red blood cells. Afterward, the blend was subjected to centrifugation at a force of 800 g for 10 min. Afterward, the pellet was suspended again using DMEM-F12. After passing through a 40 μm mesh, the liquid that had been resuspended was subjected to centrifugation at 800 g for 5 min. The adipocyte pellet was suspended again and grown in DMEM-F12 medium with 10% fetal bovine serum (FBS, Gibco, New York, NY, United States) at a temperature of 37 °C and a CO2 concentration of 5%. Mature adipocytes are formed by induction, which consists of 10% FBS, DMEM-F12, 50 μM oleic acid, 0.5 M octoic acid, 50 nM insulin, and 50 nM dexamethasone. In the course of the induction procedure, 10 μg of small extracellular vesicles were introduced into each well on day 0 and subjected to treatment for 24 h. The primary Porcine adipocytes were seeded in 12-well plates with a density of 1.0 × 105 per well. Transfection with miR-146a-5p inhibitor (40 nM) (GenePharma, Shanghai, China) was initiated using lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA) when the cell density reached 70%. Further analysis was conducted on cells harvested on day 8 of differentiation.

Isolation of small extracellular vesicle

Upon achieving 80% confluence, the cells were rinsed thrice with phosphate buffer saline (PBS, Sangon Biotech, Shanghai, China) and subsequently cultured in a fresh DMEM-F12 medium for 48 h. Ultracentrifugation was employed to isolate small extracellular vesicles from the culture supernatant. The particular procedures were as follows. After subjecting the culture supernatant to centrifugation at 2000 × g for 10 min and 12,000 × g for 30 min, the large fragments and deceased cells were eliminated. The supernatant was subjected to an ultracentrifuge at a speed of 100,000 × g for 70 min. In the end, the cells were washed in 38 ml of PBS and subjected to ultracentrifugation at 100,000 × g for 70 min. The pellets were resuspended in 100 μL of PBS. Carefully remove the supernatant, then resuspend the pellet containing small extracellular vesicles in PBS and store it at − 80 °C. The protein concentration of small extracellular vesicles was determined using the BCA Protein assay kit (Bioteke, Beijing, China).

Transmission electron microscopy analysis

After incubating 10 μL of small extracellular vesicles on formalin-coated copper for 5 min, the surplus liquid was removed. Apply uranyl acetate onto the grid for negative staining and leave it for 1 min, then remove any extra liquid. The specimens were analyzed utilizing a transmission electron microscope with an acceleration voltage of 100 kV.

Nanoparticle tracking analysis

Small extracellular vesicles were diluted with PBS to the appropriate concentration. Nanoparticle tracking analysis (NTA) was used to measure the dimensions of small extracellular vesicles obtained from porcine primary skeletal muscle stem cells. To obtain detailed guidance on how to operate the device, which includes concise explanations on how to load samples, capture photographs, and analyze results, please consult the manual.

MTT assays

MTT assays were employed to assess cell proliferation. The optimized cell number (5000 cells/well) was used to seed the cells in a 96-well plate at a density of one well per cell. Following 24 h of seeding, the cells were subjected to a dosage of 10 μg/mL of small extracellular vesicles. Each well received twenty microliters of MTT solution (5 mg/ml), which was incubated for 4 h. After that, the reaction was stopped by adding 100 μl of dimethyl sulfoxide (DMSO) and incubating at 37 °C in the absence of light for 10 min. Cell growth was evaluated by quantifying the optical density at wavelengths of 570 nm and 630 nm (Absorbance, OD570-630 nm).

Measurement of triglyceride content

The quantity of TG in adipocytes was measured utilizing an assay kit as previously explained25. In short, the cells were washed and lysed using the provided lysis buffer. Subsequently, the TG assay reagent was added as per the instructions given by the manufacturer. A spectrophotometer plate reader was used to measure the optical density of the solution at 510 nm. The TG concentration was determined using the standard curve for each assay and the results were adjusted based on the total amount of cellular protein (Biovision, Milpitas, CA, USA).

Staining with oil red O

Porcine primary adipocyte was washed with PBS, followed by fixation in 4% formaldehyde in PBS for 30 min at room temperature, and subsequently washed three times with PBS. Afterward, the cells were stained with Oil Red O (#O1391; Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Following three rinses with water, the microscope was used to observe and capture images of the lipid droplets (TE2000-E; Nikon, Tokyo, Japan).

Quantitative PCR in real-time

Total mRNA was extracted using Trizol reagent (15596-026, Thermo Fisher Scientific, Shanghai, China). Following DNase I treatment (2270A, Takara Bio, Kusatsu, Shiga, Japan), 1 μg of total RNA was converted into cDNA using MLV Reverse Transcriptase (M1705, Promega, Madison, WI, USA) and either oligo (dT) 18 primer or a dedicated stem-loop primer for miR-146a-5p. The STRATAGENE Mx3005P Sequence Detection System and SYBR Green Master Mix (Promega) were utilized for conducting real-time PCR. The findings were standardized to the levels of the GAPDH and U6 by employing the 2-ΔΔCt technique. Supplement Table 1 displays the primer sequences utilized.

Western blot analysis

The cells were disrupted using RIPA buffer which included a protease inhibitor (P7626, Sigma) at a concentration of 1 mmol/L PMSF. The BCA Protein Assay kit (Thermo Fisher Scientific, 23,227) was used to determine the concentration of the protein. According to the instructions provided by the manufacturer, the primary antibodies used were rabbit anti-Alix (1:1000, #D262028; Sangon Biotech), rabbit anti-TSG101 (1:2000, #381,538; ZEN BIO), rabbit anti-CD9 (1:1000, #AP68-965-100; Abcepta), rabbit anti-CD63 (1:2000, #D160973; Sangon Biotech), rabbit anti-PPARγ (1:1000, #2443; CST), and rabbit anti-GAPDH (1:5000, #BS65529; Bioworld). A blocking buffer was used to incubate primary antibodies overnight at 4℃. Secondary antibodies from Life Technologies were then added for 1 h. Capture detection by chemiluminescent imaging system. The Image J software was used to quantify and analyze the protein grayscales.

Statistical analyses

For one-way ANOVA, stand-alone sample t-test analysis, and plotting the statistical analysis was performed using SPSS software (version 25) and GraphPad Prism (version 9.0). The mean ± SEM was used to express all of the results. The importance of the distinction was evaluated based on a level of * p < 0.05 or ** p < 0.01. The levels of statistical significance of the difference between the groups are represented by the letters a, b, and c. Distinct letters indicate a substantial disparity, while identical letters indicate insignificance in the disparity.

Results

Viability of porcine primary adipocyte is inhibited by small extracellular vesicles derived from porcine primary skeletal muscle stem cells

We cultured the extracted porcine MuSCs in basal medium DMEM and collected the supernatant after 24 h to obtain sEV by ultracentrifugation. By observing the morphology of sEV by transmission electron microscopy (Fig. 1a) and detecting the size distribution by nanoparticle size, we found that MuSCs-derived sEV were mainly distributed around 150 nm (Fig. 1b). sEV-specific marker protein assay using Western Blot showed positive expression of Alix, TSG101, CD9, and CD63 (Fig. 1c). This indicates successful extraction of MuSCs-derived sEV. Isolated porcine primary adipocytes were inoculated and cultured, and cell viability was assayed every 12 h. We found that MuSCs-derived sEV inhibited the cell viability of porcine primary adipocyte throughout the experiment (Fig. 1d). It was shown that MuSCs-derived sEV inhibited the proliferation of porcine primary adipocyte.

Fig. 1
figure 1

Skeletal muscle stem cell-derived small extracellular vesicles inhibit porcine adipose precursor cell viability. (a) Transmission electron microscopy of sEV derived from porcine skeletal muscle stem cells demonstrated (Note: Blue arrows indicate small extracellular vesicles, scale bar = 200 nm). (b) Nanoparticle tracking analysis was used to determine the size distribution and concentration of sEV. (c) Western Blot was performed to detect sEV marker proteins Alix, TSG101, CD9, and CD63. (d) sEV-treated porcine primary adipocytes were assayed for cell viability by MTT assay (n = 6). Values are presented as means ± SEM, *P < 0.05, and **P < 0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups.

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Porcine primary skeletal muscle stem cells-derived small extracellular vesicles significantly inhibit adipogenesis

Porcine primary adipocytes were treated with MuSCs-derived sEV and induced to differentiate until cell maturity on day 8, and a reduction in lipid droplet enrichment was found in the MuSCs-derived sEV group using oil red O staining (Fig. 2a). The control group exhibited noticeably elevated levels of triglycerides (TG) compared to the sEV group derived from MuSCs, as indicated by TG assays (Fig. 2b). By isolating RNA and proteins to identify associated genes, we observed a significant decrease in the expression of distinct polyester-related genes PPARγ, C/EBPα, CD36, FABP4, and ACC in the sEV group derived from MuSCs compared to the control group. Additionally, IL-6 exhibited a significant increase (Fig. 2c–h). IL-6 is a pro-inflammatory factor that inhibits cellular uptake of free fatty acids. Meanwhile, Western Blot results showed that MuSCs-derived sEV groups inhibited the protein synthesis of PPARγ, with similar results obtained with qPCR (Fig. 2i,j). The aforementioned findings suggest that the adipocytes’ capacity for differentiated polyester was suppressed following treatment with sEV derived from MuSCs.

Fig. 2
figure 2

Skeletal muscle stem cells-derived small extracellular vesicles significantly inhibit adipogenesis. (a) Oil Red O staining of porcine primary adipocytes treated with PBS (CON) or sEV (scale bar = 50 μm). (b) Statistical results of TG content of porcine primary adipocytes treated with PBS or sEV (n = 4). (c–h) Real-time quantitative PCR assay of adipogenesis-related genes PPARγ, C/EBPα, fatty acid synthesis related gene CD36, FABP4, ACC, and pro-inflammatory factor IL-6 in porcine primary adipocytes treated with sEV (n = 6). (i, j) Gray-scale results of PPARγ protein expression by Western Blot after sEV treatment of porcine primary adipocytes (n = 6). Values are presented as means ± SEM, *P < 0.05, and **P < 0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups.

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Insulin or oleic acid treatment of MuSCs-derived sEV reverses the inhibition of adipogenesis

Normal MuSCs-derived sEV has an inhibitory effect on adipocyte differentiation, whereas INS and OA, as inducers of adipocyte differentiation, have a promotional effect on adipocyte differentiation, and can sEV secreted by INS- or OA-treated MuSCs affect adipocyte differentiation phenotypes? MuSCs were treated with INS and OA, and after 24 h, cell supernatants were collected to extract sEV, named sEV + INS and sEV + OA, respectively, and treated with porcine primary adipocyte, induced to differentiate to cellular maturity, and subsequently stained with Oil Red O. It was observed that the number of lipid droplets in the control cells exceeded that of the sEV-treated group, and the sEV-treated adipocytes treated with sEV + INS and sEV + OA also exhibited a higher number of lipid droplets compared to the sEV-treated group (Fig. 3a). The sEV-treated group showed a notable reduction in TG content compared to the control group, while the sEV group treated with INS and OA exhibited a significant increase in TG content (Fig. 3b). In the sEV-treated group, the qPCR results indicated a significant decrease in the expression of polyester-related genes PPARγ, C/EBPα, CD36, FABP4, and ACC, while IL-6 showed a significant increase compared to the control group. However, the sEV group treated with INS and OA exhibited restoration of PPARγ, C/EBPα, CD36, FABP4, ACC, and IL-6 levels to normal or even higher levels (Fig. 3c–h). The western blot findings additionally confirmed that sEV suppressed the protein quantities of PPARγ, whereas adipocytes treated with sEV + INS and sEV + OA exhibited a notable increase in the level of PPARγ protein (Fig. 3i,j). The results indicate that INS and OA treatment of MuSCs may have altered the heterogeneity of sEV and thus reversed the inhibitory effect on adipocytes.

Fig. 3
figure 3

Insulin or oleic acid treatment of MuSCs-derived sEV reverses the inhibition of adipogenesis. (a) Oil Red O staining of porcine primary adipocytes treated with PBS (CON), sEV, sEV + INS, and sEV + OA (scale bar = 50 μm). (b) TG contents of differentiated porcine primary adipocytes treated with PBS, sEV, sEV + INS, and sEV + OA (n = 6). (c–h) Real-time quantitative PCR detection of adipogenesis-related genes PPARγ, C/EBPα, and fatty acid synthesis-related gene CD36, FABP4 ACC, and pro-inflammatory factor IL-6 in porcine primary adipocytes treated with PBS, sEV, sEV + INS, and sEV + OA (n = 12). (i, j) Western Blot results of adipogenesis-related gene PPARγ in porcine precursor adipocytes (n = 4). The results were presented as mean ± standard error of the mean (SEM). The letters a, b, and c represent the level of statistical significance of the difference between the groups. Different letters mean a significant difference, and the same letters mean the difference is not significant.

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MuSCs-derived sEV miR-146a-5p is a key microRNAs that inhibits adipocyte differentiation

Treatment of MuSCs with modulators such as INS and OA may lead to changes in the composition of secreted sEV, which in turn regulate adipocyte differentiation through the sEV-mediated pathway. Fresh MuSCs-derived sEV and porcine primary adipocyte-derived sEV were extracted for high-throughput assay26, and our results demonstrated that MuSCs-derived sEV were more than twofold more different from porcine primary adipocyte-derived sEV in the top 4 miRNAs (Fig. 4a). To confirm the precision of the sequencing outcomes, MuSCs-derived sEV that were exposed to INS and OA were subsequently treated with porcine primary adipocyte and then prompted to undergo differentiation until reaching cell maturation on day 8 to gather the plate. Subsequently, total RNA was extracted to examine the expression of pertinent genes. Confirmed for miR-146a-5p, miR-129a, miR-9–3, and miR-30e. After 8 days of differentiation, the miR-146a-5p expression in the sEV derived from MuSCs was notably elevated in comparison to the other groups (Fig. 4b). In previous investigations conducted in our research facility, it was discovered that miR-146a-5p additionally impeded the process of adipocyte differentiation27. The findings indicate that miR-146a-5p could potentially serve as a significant signaling molecule in sEV derived from MuSCs, playing a role in the process of adipogenesis.

Fig. 4
figure 4

MuSCs-derived sEV miR-146a-5p is a key gene that inhibits adipocyte differentiation. (a) High-throughput screening and comparison of miRNA expression abundance in adipocyte-derived sEV and MuSCs-derived sEV. (b) After treatment with PBS, sEV, sEV + INS, and sEV + OA, differentiated porcine precursor adipocytes were induced to mature, and the expression levels of miR-146a-5p, miR-129a, miR-9–3, and miR-30e were detected by real-time quantitative PCR (n = 6). The results were presented as mean ± standard error of the mean (SEM). The letters a and b, represent the level of statistical significance of the difference between the groups. Different letters mean a significant difference, and the same letters mean the difference is not significant.

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The molecule miR-146a-5p, which is abundant in sEV derived from MuSCs, plays a crucial role in suppressing the differentiation of adipocytes

The aforementioned findings indicate that miR-146a-5p could potentially serve as a significant signaling molecule present in sEV derived from MuSCs, playing a role in the process of adipogenesis. Subsequently, we simultaneously treated porcine primary adipocyte and initiated differentiation by employing miR-146a-5p INC and miR-146a-5p inhibitor alongside MuSCs-derived sEV. The results indicate that sEV effectively reduced the accumulation of fat droplets, the amount of TG, and the expression of genes associated with adipogenesis such as PPARγ, C/EBPα, CD36, FABP4, and IL-6 in adipocytes when compared to the control group (CON). However, the inhibitory effect was weakened when the miR-146a-5p inhibitor was co-incubated with sEVs (sEV + inhibitor) (Fig. 5a–i). These results suggest that MuSCs-derived sEV may mediate and inhibit the differentiation of porcine primary adipocyte by delivering miR-146a-5p.

Fig. 5
figure 5

miR-146a-5p enriched in MuSCs-derived sEV is a key molecule inhibiting adipocyte differentiation. (a) After treatment of sEV and transfections of INC (control) or miR-146a-5p inhibitor, the maturation of porcine precursor adipocytes was induced, and oil red O staining was performed (scale bar = 50 μm). (b) After sEV were processed and transfected with INC or inhibitor, porcine precursor adipocytes were induced to mature, and TG content was detected (n = 6). (c–g) After sEV were processed and transfected with INC or inhibitor, porcine precursor adipocytes were induced to mature, and the adipogenesis-related genes PPARγ, C/EBPα, and fatty acid synthesis-related gene CD36, FABP4, and pro-inflammatory factor IL-6 was detected by real-time quantitative PCR (n = 6). (h, i) After sEV were processed and transfected with INC or inhibitor, porcine precursor adipocytes were induced to mature, and adipogenesis-related gene PPARγ was detected by Western Blot (n = 6). The letters a and b, represent the level of statistical significance of the difference between the groups. Different letters mean a significant difference, and the same letters mean the difference is not significant.

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Discussion

The pork industry aims to achieve a harmonious balance between growth performance, carcass yield, meat quality, and welfare-friendly production to maximize its economic advantage28. The livestock and meat sector are always searching for fresh options to enhance animal performance and enhance carcass traits. This, along with the consumer demand for leaner pork, has led to the development of high-lean breeds29. Therefore, how to improve the quality of pork has always been the focus of research in China, in the pursuit of a high lean meat ratio at the same time also requires better color and flavor, and the key to improving the quality of pork is still the development of muscle and fat. It has been shown that adipose deposition is mainly dependent on the proliferation, differentiation, and polyester of adipose precursor cells and that this differentiation process is regulated by complex gene transcripts, such as PPARγ, C/EBPα & SREBP-1, etc., in which triglyceride content is also key to adipose development30,31. Indeed, the interaction between muscle and fat has not been extensively studied to date.

There are many mechanisms of dialog between muscle and fat, and since they are both derived from the mesoderm, they are interdependent as early as the onset of life. As the organism grows and develops, the interrelationship between the two becomes even closer. It has been reported that fats from different tissue sources can act heterologously and dynamically on skeletal muscle32. Muscle paracrine factors may hinder the differentiation of intramuscular adipose precursor cells, as indicated by available evidence. Following the co-cultivation of muscle cells and adipocytes and the subsequent induction of adipocyte maturation, a notable reduction in adipocyte triglyceride levels was observed33. Additionally, the outcomes of oil red O staining suggested an inhibition in adipocyte differentiation34. Furthermore, the expression of polyester-associated genes, namely PPARγ and C/EBPα, exhibited a significant decrease35. Nevertheless, there is no report on the involvement of sEV in the regulation of muscle and fat interactions in pigs. Hence, this research involved the extraction of sEV from MuSCs of porcine skeletal muscle. These sEVs were then utilized for the treatment of adipocytes, resulting in the inhibition of porcine primary adipocyte’ maturation. This inhibition was confirmed through triglyceride assay and Oil Red O staining. Additionally, qPCR and western blot analysis demonstrated the suppression of adipocyte differentiation, as evidenced by the significant decrease in PPARγ, C/EBPα, CD36, FABP4, and ACC polyester-related genes.

Insulin and OA are important factors in inducing adipocyte differentiation and maturation. Pancreatic β-cells secrete insulin, a protein hormone that plays a crucial role in maintaining glucose balance and metabolism. Additionally, insulin stimulates the increase of triglycerides (TG) in adipocytes36,37. Our research has earlier demonstrated that miR-146a-5p suppresses fat cell formation by targeting the insulin receptor (IR). Additionally, miR-146a-5p was found to reduce tyrosine phosphorylation of IRS-1, indicating its significant role in insulin signaling42. OA usually exists in the form of TG, and it has been found that the addition of appropriate amounts of OA during adipocyte differentiation can elevate triglyceride concentration and PPARγ expression38. In our previous study, we found that exosomal miR-146a-5p from skeletal muscle inhibits adipogenesis by targeting GDF5-PPAR signaling, and miR-146a-5p inhibitor reversed this inhibitory phenotype27. In this current research, the treatment of sEV derived from skeletal muscle satellite cells with insulin or OA-induced differentiation of porcine primary adipocyte to maturity. By triglyceride assay and oil red O staining, it was found that the polyester capacity of adipocytes was reversed and significantly enhanced, and the expression of polyester-related genes was significantly increased in comparison with the normal sEV-treated group.

MicroRNAs play a crucial role in various biological processes, such as cell proliferation, programmed cell death, and cell differentiation. In recent years, there have been reports on the involvement of the miR-146 family, specifically miR-146b and miR-146a, in life activities such as rheumatoid arthritis, esophageal cancer, ovarian cancer, hepatic cancer, and aging, and studies related to glucose-lipid metabolism have begun to be reported in the last few years39,40. Still, there have been fewer studies on the miR-146 family in porcine adipogenesis. The study conducted by Sun et al. demonstrated that miR-146 could potentially be used as a therapy to reduce skeletal muscle fibrosis following an injury41. Previous studies in our laboratory found that differentiation of porcine adipocytes was inhibited after treatment with TNF-α, and this effect was found to be mediated through miR-146a-5p42. According to Zhang et al., the research discovered that miR-146a-5p targeted TRAF6 and hindered the differentiation of porcine intramuscular preadipocytes by controlling the AKT/mTORC1 signaling pathway43. The study by Wang et al. revealed that the increase in miR-146a-5p expression was linked to the differentiation of 3T3-L1 cells. Additionally, miR-146a-5p facilitated the differentiation of 3T3-L1 cells by targeting the ErbB4 and ERK1/2/PPAR-γ signaling pathways44. The miRNA high-throughput sequencing revealed a significant increase of miR-146a-5p in sEV derived from MuSCs compared to sEV derived from adipocytes in this study. The combination of porcine primary adipocyte by miR-146a-5p inhibitor and sEV derived from MuSCs showed that inhibiting miR-146a-5p no longer prevented adipocyte differentiation, indicating that miR-146a-5p could play a crucial role in the inhibition of adipocyte differentiation polyester by sEV from MuSCs.

Conclusion

This study used a high-throughput screening technique to reveal that miRNAs in muscle stem cells-derived sEV and primary adipocytes-derived sEV from long white piglets were differentially screened and functionally characterized. The findings suggest that heterogeneity variations in porcine muscle-derived sEV fractions are closely associated with miR-146a-5p and control adipogenesis by influencing miR-146a-5p alterations. The results offer a scientific foundation for the involvement of sEV in the exchange of communication between muscle and fat, leading to enhanced meat quality.