Aerobic exercise regulates macrophage polarization and improves insulin resistance in mice: the mediating role of miR-221-3p

doi:10.12122/j.issn.1673-4254.2026.01.08

Abstract

Abstract:

Objective To explore the role of miR-221-3p in mediating the positive effects of aerobic exercise on macrophage polarization in the adipose tissues and insulin resistance (IR). Methods Sixteen normal C57BL/6J mice and 16 mice with IR induced by high-fat diet (HFD) feeding for 12 weeks were both randomized into sedentary group and exercise group with aerobic exercise training on a treadmill (5 times per week for 8 consecutive weeks). All the mice were examined for changes in body weight, body composition, fasting blood glucose, blood lipid levels, insulin levels, miR-221-3p expression level, mRNA levels of Socs1, Tnf-α and Arg-1, and protein levels of SOCS1, JAK1, p-STAT1, and p-STAT3 in the adipose tissues, and the targeting relationship between miR-221-3p and SOCS1 was validated using dual-luciferase reporter gene assay. In RAW264.7 macrophages, the effects of transfection with miR-221-3p mimic or inhibitor on macrophage polarization were observed. Results In mice with normal feeding, aerobic exercise significantly decreased body weight, fat mass, fat percent, fasting blood glucose, serum insulin level, HOMA-IR, and TC and TG levels, and reduced miR-221-3p levels in both the plasma and the adipose tissues. The sedentary IR mice showed significantly increased miR-221-3p levels in both the plasma and adipose tissue, increased protein levels of iNOS, JAK1, and p-STAT1/STAT1, and decreased protein levels of Arg-1, SOCS1 and p-STAT3/STAT3, which were significantly reversed after aerobic exercise intervention. Dual-luciferase reporter gene assays validated the targeting relationship between miR-221-3p and SOCS1. In RAW264.7 macrophages, miR-221-3p overexpression significantly reduced Socs1 and Arg-1 mRNA expression, whereas miR-221-3p inhibition obviously promoted M2 polarization of the macrophages. Conclusion Aerobic exercise improves HFD-induced IR in mice possibly by inhibiting miR-221-3p to activate the SOCS1 and JAK/STAT signaling pathway, thereby promoting macrophage M2 polarization and alleviating chronic inflammation in the adipose tissue.

Key words: aerobic exercise, insulin resistance, microRNA, macrophage polarization

Nan LI, Liang ZHANG, Qiaofeng GUO, Yue ZHOU, Changjiang LIU. Aerobic exercise regulates macrophage polarization and improves insulin resistance in mice: the mediating role of miR-221-3p[J]. Journal of Southern Medical University, 2026, 46(1): 74-82.

Figures/Tables 9

Tab.1 Primers sequences for RT-qPCR

Gene	Primer sequences (5'-3')
miR-221-3p	RT	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGAAACCCA
miR-221-3p	F	ACACTCCAGCTGGGAGCTACATTGTCTGC
U6	F	CTCGCTTCGGCAGCACA
U6	R	AACGCTTCACGAATTTGCGT
Tnf-α	F	CCCTCACACTCAGATCATCTTCT
Tnf-α	R	GCTACGACGTGGGCTACAG
Arg-1	F	ACAGCAGAGGAGGTGAAGAGTAC
Arg-1	R	AGTCAGTCCCTGGCTTATGGT
Socs1	F	CTGCGGCTTCTATTGGGGAC
Socs1	R	AAAAGGCAGTCGAAGGTCTCG
β-actin	F	GTGCTATGTTGCTCTAGACTTCG
β-actin	R	ATGCCACAGGATTCCATACC

Fig.1 Changes in body weight of the mice in each group (n=6). A: Changes in body weight. B: Changes in fat mass. C: Changes in lean body mass. D: Changes in fat percent. CS: Normally fed sedentary group without exercise; CE: Normally fed sedentary group with aerobic exercise; HS: High-fat diet sedentary group; HE: High-fat diet group with aerobic exercise. **P<0.01 vs CS group, ##P<0.01 vs HS group.

Tab.2 Changes in the indicators of the mice in each group

Parameter	CS	CE	HS	HE
Fasting blood glucose (mmol/L)	6.70±0.38	6.13±0.44	13.10±0.99**	7.56±0.69^##
Insulin (μIU/mL)	7.74±0.62	8.27±2.70	24.73±2.41**	11.70±3.30**^##
HOMA-IR	2.32±0.22	2.24±0.72	14.46±2.27**	3.87±0.84*^##
TC (mmol/L)	3.08±0.13	3.02±0.53	4.67±0.40**	3.90±0.70**^##
TG (mmol/L)	0.59±0.07	0.60±0.09	0.88±0.19**	0.72±0.07^##
HDL (mmol/L)	2.87±0.47	2.86±0.27	3.76±0.73**	3.56±0.23
LDL (mmol/L)	0.15±0.08	0.14±0.06	0.32±0.13	0.31±0.13

Fig.2 Changes in miR-221-3p levels in each group. A: Expression levels of miR-221-3p in plasma (n=6). B: Expression levels of miR-221-3p in adipose tissue (AT). **P<0.01 vs CS group, ##P<0.01 vs HS group.

Fig.3 Targeting relationship between Socs1 and miR-221-3p (A) and dual-luciferase activity expression (B). n=3, **P<0.01 vs WT group.

Fig.4 Relative mRNA levels of miR-221-3p (A) and Socs1 (B) enriched in RAW 264.7 cells (n=3). **P<0.01 vs NC group, ##P<0.01 vs mimic NC group, $$P<0.01 vs inhibitor NC group.

Fig.5 Relative mRNA levels of Tnf-α and Arg-1 enriched in RAW 264.7 cells (n=3). *P<0.05, **P<0.01 vs NC group, #P<0.05, ##P<0.01 vs mimic NC group, $$P<0.01 vs inhibitor NC group.

Fig.6 Changes of macrophage polarization-related proteins in the adipose tissue of mice after aerobic exercise intervention. A: Expression of iNOS proteins in adipose tissue. B: Expression of Arg-1 proteins in adipose tissue. n=6. *P<0.05, **P<0.01 vs CS group, #P<0.05, ##P<0.01 vs HS group.

Fig.7 Comparison of the expression levels of JAK/STAT pathway proteins SOCS1 (A), JAK1 (B), p-STAT1/STAT1 (C), and p-STAT3/STAT3 (D) in the adipose tissue of the mice. n=6. **P<0.01 vs CS group, #P<0.05, ##P<0.01 vs HS group.

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