Exosomes from folic acid-treated subpatellar fat pad-derived mesenchymal stem cells promote M2 polarization of macrophages in vitro

doi:10.12122/j.issn.1673-4254.2026.01.18

Abstract

Abstract:

Objective To evaluate the effect of exosomes derived from folic acid (FA)-treated infrapatellar fat pad mesenchymal stem cells (IPFP-MSCs) on M1 and M2 polarization of macrophages in vitro. Methods Infrapatellar fat pad tissues were obtained from surgical patients without knee osteoarthritis to isolate IPFP-MSCs. The exosomes were extracted from the cell cultures with or without FA treatment and identified by transmission electron microscopy, TEM, NTA and Western blotting. RAW264.7 cells were induced with lipopolysaccharide (LPS) and incubated with exosomes from FA-treated or untreated IPFP-MSCs for 12 h, and Exos uptake was observed using confocal microscopy. The changes in expression levels of IL-1β, IL-6, TNF-α, iNOS, ARG1, MRC1, and CD206 in the macrophages were detected using qRT-PCR, ELISA, flow cytometry and immunofluorescence staining. Results The exosomes derived from IPFP-MSCs showed a typical cup shape, were positive for CD9 and CD81, and could be uptaken by macrophages. In LPS-induced macrophages, incubation with exosomes from FA-treated IPFP-MSCs significantly decreased the expressions of IL-1β, IL-6, TNF‑α, and NOS2, and increased the expressions of ARG1 and MRC1. Treatment of the macrophages with exosomes from FA-treated IPFP-MSCs significantly lowered CD86-positive cell percentage, increased CD206-positive cells and the CD206/CD86 ratio, lowered cellular expression of iNOS, and enhanced the expression of CD206. Conclusion Exosomes from FA-treated IPFP-MSCs promotes M2 polarization of macrophages more effectively than exosomes from unmodified IPFP-MSCs, suggesting a new exosome modification strategy for targeted treatment of knee osteoarthritis.

Key words: folic acid, macrophage, exosomes, mesenchymal stem cells, osteoarthritis

Zhe WANG, Keyu KONG, Minghao JIN, Sonu NG, Wenxuan FAN, Zanjing ZHAI, Zihao HU, Lin NIU, Yansong QI, Yongsheng XU. Exosomes from folic acid-treated subpatellar fat pad-derived mesenchymal stem cells promote M2 polarization of macrophages in vitro[J]. Journal of Southern Medical University, 2026, 46(1): 166-174.

Figures/Tables 7

Tab.1 Primer sequence for qRT-PCR

Gene	Forward (5′-3′)	Reverse (5′-3′)
ARG1	CCACAGTCTGGCAGTTGGAAG	GGTTGTCAGGGGAGTGTTGATG
MRC1	GGCTGATTACGAGCAGTGGA	CATCACTCCAGGTGAACCCC
TNF-α	GCCTCTTCTCATTCCTGCTTGTGG	GTGGTTTGTGAGTGTGAGGGTCT
IL-1β	TCGCAGCAGCACATCAACAAGAG	AGGTCCACGGGAAAGACACAGG
NOS2	ACTCAGCCAAGCCCTCACCTAC	TCCAATCTCTGCCTATCCGTCTCG
IL-6	CTTCTTGGGACTGATGCTGGTGAC	AGGTCTGTTGGGAGTGGTATCCTC
GADPH	GGCAAGTTCAACGGCACAG	CGCCAGTAGACTCCACGACAT

Fig.1 Identification of infrapatellar fat pad mesenchymal stem cells (IPFP-MSCs) by flow cytometry (A-H) and under optical microscope (I).

Fig.2 Identification of exosomes from IPFP-MSCs (IPFP-Exos) and its distribution in RAW264.7 macrophages. A: TEM observation showing cup-shaped IPFP-Exos. B: NTA showing homogenous diameter of the vesicles. C: Positive expression of surface markers CD9 and CD81 on IPFP-Exos; D: Confocal microscopy for observing distribution of IPFP-Exos in RAW264.7 macrophages.

Fig.3 Changes in the mRNA expression levels of IL-1β (A), IL-6 (B), TNF-α (C), NOS2(D), ARG1 (E) and MRC1 (F) (n=3). **P<0.01, ****P<0.0001 vs LPS group.

Fig.4 Results of ELISA for detecting expression levels of IL-1β (A), IL-6 (B) and TNF-α (C) in the cell supernatant (n=5). ****P<0.0001 vs LPS group.

Fig.5 Expressions of CD86 and CD206 in RAW264.7 macrophages incubated with the exosomes detected by flow cytometry. A: Two-dimensional density maps of flow cytometry showing the expression of M1-type marker CD86 and M2-type marker CD206 in different groups. B: Bar chart of the percentage of CD86-positive cells in different groups. C: Bar chart of the percentage of CD206-positive cells in different groups. D: The ratio of CD206/CD86 in different groups. (n=4/5). ***P<0.001, ****P<0.0001 vs LPS group.

Fig.6 Immunofluorescence staining of iNOS and CD206 in RAW264.7 macrophages incubated with the exosomes. A: Expression of iNOS in RAW264.7 cells in different treatment groups. B: Statistical analysis of the proportion of iNOS-positive cells in each group. C: Expression of CD206 in RAW264.7 cells in different groups. D: Statistical analysis of the fluorescence intensity of CD206 in each group (n=3). **P<0.01, ***P<0.001, ****P<0.0001 vs LPS group.

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