2.1 Preparation of P-PRP and L-PRP

Fasting venous blood was collected from two male healthy volunteers (age 25 and 37) in the Department of Blood Transfusion, the Second Affiliated Hospital of Guilin Medical College after signing informed consent. The P-PRP and L-PRP were prepared using the PRP preparation kits (VEGO, China) and based on the described methods [31]. The concentrations of platelets and WBCs were determined by a blood analyzer. The collected P-PRP and L-PRP were preserved in −80°C. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Guilin Medical College.

2.2 Isolation and Identification of Exosomes

The P-PRP and L-PRP samples were centrifuged at 500g for 10 min at 4°C. Then, the supernatant was centrifuged at 2000g for 30 min, and the supernatant was collected for another 10,000g for 30 min using a centrifuge (5810R, Eppendorf, Hamburg, Germany). Afterward, the supernatant was harvested to a high-speed centrifuge tube, and the volume was replenished with sterile PBS to 20 mL, and then was centrifuged at 120,000g for 70 min at 4°C using a high-speed centrifuge (Optima XPN-100, Beckman, Brea, CA, USA) to obtain high-purity exosomes. The harvested exosomes were identified by CD63, TSG101, and CD9 markers using Western blot. The morphology and size distribution were detected utilizing transmission electron microscopy (TEM) (JOEL Ltd, Japan) and nanoparticle tracking analysis (NTA) (Malvern, UK), respectively.

2.3 Cell Culture

THP-1 cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). All cells were maintained in RPMI 1640 medium with 10% FBS and 1% streptomycin/penicillin (Gibco, Waltham, MA, USA) at 37°C with 5% CO₂. In indicated assays, THP-1 cells were incubated with 200 ng/mL phorbol-12-myristate-13 acetate (PMA) to induce differentiation.

2.4 Determining the Cellular Uptake of Exosomes

Exosomes were incubated with PKH67 (Thermo Fisher, USA) for 5 min at room temperature, and 1% BSA was used to terminate staining. The free dye was removed, and PKH67-stained exosomes were collected by centrifugation at 12,000g for 90 min.

THP-1 cells were harvested, seeded in 24-well plates, and induced into macrophages using PMA. Cells were then cultured in RPMI 1640 medium without serum. After 24 h, cells were incubated with 10 μL of PKH67-stained exosomes for 24 h. Afterward cells were fixed in 4% paraformaldehyde, treated with 0.1% TritonX-100, blocked with 3% BSA, and stained with Mounting Medium containing DAPI. Finally, cells were observed and photographed under confocal laser scanning microscopy.

2.5 Cell Counting Kit-8 (CCK-8) Assay

After THP-1 cells were collected, cells were added to RPMI 1640 medium with 10% exosome-free serum and 200 ng/mL PMA. Afterward cells were seeded into 96-well plates (1 × 10⁴ cells/well) for 24 h cultivation. Then, cells were mixed with different concentrations of exosomes (final concentrations of 5, 10, 20 and 50 μg/mL) for incubation, and the control cells were PMA-induced THP-1 cells treated with PBS. At 24, 48, and 72 h, 10 μL CCK-8 (Beyotime, Shanghai, China) was added to each well and treated for 1 h. Optical density values at wavelength 450 nm were tested by a microplate reader (Bio-Rad, USA).

2.6 Cell Apoptosis

Cell apoptosis was evaluated utilizing Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, CA, USA). After THP-1 cells were collected, cells were added to RPMI 1640 medium with 10% exosome-free serum and 200 ng/mL PMA. Cells were seeded into 24-well plates (1 × 10⁵ cells/well) for 24 h cultivation. Then, cells were mixed with exosomes (final concentration: 20 μg/mL) for 72 h incubation. After that, THP-1 cells (1 × 10⁵) were harvested and resuspended in the binding buffer. Then, cells were dyed with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) for 15 min without light at room temperature. The apoptotic cells were determined by the FACSCalibur flow cytometer (BD Biosciences, USA).

2.7 Western blot

Total proteins were collected using RIPA and quantitated by the BCA method. Proteins were run on the SDS-PAGE and transferred to PVDF membranes. After hindered in 5% skim milk, the membranes were probed to anti-TSG101 (1:1000, 28283-1-AP), anti-CD63 (1:1000, 25682-1-AP), anti-CD9 (1:1000, 20597-1-AP), anti-CD206 (1:1000, 18704-1-AP), anti-TGF-β1 (1:500, sc-130348), anti-IL-1β (1:1000, AF5103), anti-TNF-α (1:1000, 60291-1-Ig), and GAPDH (1:10000, 60004-1-Ig) for 12 h and treated with second antibody IgG. All primary antibodies were acquired from Proteintech (Proteintech, Wuhan, China) except for TGP-β1 (Santa Cruz, CA, USA) and IL-1β (Affinity, Jiangsu, China). The bands were detected utilizing an ECL system (Bio-Rad, USA) and analyzed using Image J.

2.8 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNAs were collected using TransZol Up and utilized to generate cDNA by the First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech, Beijing, China). Following the supplier's protocol, the qPCR assay was carried out using the PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China). Primers used in this study are listed in Table 1. The relative expression of genes was assessed using the $${2}^{ \mbox{-} \unicode{x02206}\unicode{x02206}{C}_{{\rm{t}}}}$$ via normalizing to GAPDH.

2.9 Nitric Oxide (NO) Detection

After THP-1 cells were collected, cells were added to RPMI 1640 medium with 10% exosome-free serum and 200 ng/mL PMA for 24 h. Cells were then mixed with exosomes (final concentration: 10 μg/mL) for 72 h incubation. After that, the cell supernatant and cells were harvested and applied to detect the NO level using the Total Nitric Oxide Assay Kit (Beyotime, Shanghai, China) following the supplier's protocol. Optical density values at 540 nm were tested by a microplate reader (Bio-Rad, USA).

2.10 Statistical Analysis

All data from at least three replicates were described as mean ± SD, and data analysis was completed by GraphPad Prism 8.4.2. Comparison among multiple groups was assessed by one-way ANOVA with Tukey's post hoc test. p < 0.05 was identified as the criteria of statistically significance.

3.1 Characterization of Exosomes Derived From P-PRP and L-PRP

To clarify the action of PRP-derived exosomes on macrophages, the exosomes were first prepared from P-PRP and L-PRP, respectively. TEM results found that the extracted exosomes from P-PRP and L-PRP exhibited round or elliptical morphology with a complete double-membrane structure (Figure 1A). Besides, the NTA results demonstrated that the diameters of exosomes from P-PRP and L-PRP were approximately 155 nm (Figure 1B). Furthermore, the presence of TSG101, CD63, and CD9 in the exosomes was confirmed (Figure 1C). Therefore, these results indicated that exosomes were successfully isolated from PRP.

3.2 Coculture of Exosomes From P-PRP/L-PRP and Macrophages

To explore whether macrophages could absorb the PRP-derived exosomes, the macrophages were induced using THP-1 cells by PMA. Results showed that THP-1 cells appear to adhere to the wall and produce antennae after induced by PMA (Figure 2A), which indicated that macrophages were successfully induced. After that, internalization of PKH67-labeled exosomes was observed after coculture of the exosomes and macrophages for 24 h. It was found that the exosomes from P-PRP and L-PRP were internalized by macrophages (Figure 2B). Thus, these results suggested that the exosomes from P-PRP and L-PRPR could be taken up by macrophages.

3.3 Exosomes From L-PRP Restrained Viability and Induced Apoptosis of Macrophages

To elaborate the action of PRP-derived exosomes on macrophages, cell viability and apoptosis ability of macrophages were determined after treated by exosomes from P-PRP and L-PRP. Results showed that the exosomes from P-PRP had no significant effect on the viability of macrophages at different times (24, 48, 72 h) and concentrations (5, 10, 20, 50 μg/mL) (Figure 3A). However, the exosomes from L-PRP (20 and 50 μg/mL) significantly suppressed the viability of macrophages after treated for 48 and 72 h (p < 0.05, Figure 3A). According to this result, 20 μg/mL of exosomes with 72 h treatment was selected for subsequent experiments. Furthermore, flow cytometry results found that the exosomes from L-PRP significantly promoted the apoptosis of macrophages (p < 0.01). In contrast, exosomes from P-PRP did not affect the apoptosis of macrophages (Figure 3B). Hence, these results revealed that exosomes from L-PRP restrained viability and induced apoptosis of macrophages.

3.4 Exosomes From P-PRP Promoted M2 Polarization, and Exosomes From L-PRP Promoted M1 Polarization

To further investigate the action of PRP-derived exosomes on macrophages, the role of exosomes in macrophage polarization was detected by testing the levels of M1 markers (IL-6, IL-1β, TNF-α, IL-12, CD86, iNOS, MCP-1) and M2 markers (CD206, CD163, TGF-β1, IL-10, Arg-1). Results showed that the exosomes from P-PRP significantly enhanced the mRNA expressions of CD206 and TGF-β1 (p < 0.05, Figure 4A). However, the exosomes from L-PRP significantly elevated the mRNA expressions of IL-1β and TNF-α (p < 0.05, Figure 4A). Besides, western blot results found that the exosomes from P-PRP increased the protein levels of CD206 and TGF-β1, and the protein levels of IL-1β and TNF-α were elevated by exosomes from L-PRP (p < 0.05, Figure 4B). These results indicated that exosomes from P-PRP promoted M2 polarization, and exosomes from L-PRP promoted M1 polarization.

3.5 Exosomes From -PRP Promoted No Generation of Macrophages

To deeply explore the effect of PRP-derived exosomes on macrophages, the NO generation of macrophages was determined after treated by exosomes. Results showed that the exosomes from P-PRP had no significant effect on the NO production in macrophages and supernatant (Figure 5). Nevertheless, the exosomes from L-PRP obviously strengthened the NO generation both in macrophages and supernatant (Figure 5). In brief, these results suggested that exosomes from L-PRP promoted NO generation of macrophages.