Acacetin ameliorates insulin resistance in obesity mice through regulating Treg/Th17 balance via MiR-23b-3p/NEU1 Axis

Abstract

Background: The role of miR-23b-3p in insulin resistance (IR) remained poorly understood.

Methods: After acacetin injection, obesity-induced IR model was constructed with or without miR-23b-3p upregulation and Neuraminidase 1 (NEU1) overexpression in mice. Body weight, serum metabolite and fat percent of the mice were measured. Tests on oral glucose and insulin tolerance were performed, and inflammatory cytokines C-reactive protein (CRP), Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein 1 (MCP1) levels were quantified with enzyme-linked immunosorbent assay (ELISA). The binding sites between miR-23b-3p and NEU1 were predicted by TargetScan, and verified using dual-luciferase reporter assay. Relative expressions were detected with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Proportion of Treg and Th17 cells in total CD4⁺ T cells was detected with flow cytometry.

Results: MiR-23b-3p offset the effects of acacetin on body weight, fat percent, inflammatory cytokines levels and expressions of markers of regulatory T cells (Treg cells) and T helper 17 cells (Th17 cells), NEU1 and miR-23b-3p. NEU1 was a target of miR-23b-3p, and overexpressed NEU1 reversed the effects of upregulated miR-23b-3p on reducing Treg cells but increased body weight, fat percent and inflammatory cytokines levels, percentage of Th17 cells, and upregulated NEU1 expression.

Conclusion: Upregulation of miR-23b-3p offset the effects of acacetin on obesity-induced IR through regulating Treg/Th17 cell balance via targeting NEU1.The present findings provide a possible prevention strategy for obesity-induced IR.

Keywords: Acacetin; Inflammation; Insulin resistance; MiR-23b-3p; Neuraminidase 1; Obesity; T cells/T helper 17 cells.

Fig. 1

MiR-23b-3p upregulation promoted the effects of obesity-induced IR of mice. a Body weight of mice after obesity-induced IR model construction, Acacetin injection and miR-23b-3p upregulation was measured. b Fat percent of mice was calculated after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation. c-d Levels of fasting blood glucose (c) and fasting insulin (d) after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation was determined. e-f AUC of mice for OGTT (e) and ITT (f) after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation were quantified. g-j Levels of inflammatory cytokines CRP (g), IL-6 (h), TNF-α (i) and MCP1 (j) after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation were detected with ELISA. k Relative expressions of IL-17 and Foxp3 after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation were determined using qRT-PCR. GAPDH was used as internal control. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, vs. Control; ^{^}P < 0.05, ^{^^}P < 0.01, ^{^^^}P < 0.001, vs. Model; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. Acacetin+mimic control (MC). miR-23b-3p: MicroRNA-23b-3p; AUC: Area under the Curve; OGTT: Oral glucose tolerance test; ITT: Insulin tolerance test; CRP: C-reactive protein; IL-6: Interleukin-6; TNF-α: tumor necrosis factor-α; MCP1: monocyte chemoattractant protein 1; ELISA: enzyme-linked immunosorbent assay; Foxp3: Forkhead Box P3; qRT-PCR: quantitative real-time polymerase chain reaction

Fig. 2

NEU1 was the target of miR-23b-3p, and miR-23b-3p upregulation further enhanced the effects of obesity on NEU1 expression. a Sequences of NEU1-WT (top), miR-23b-3p (middle) and NEU1-MUT (below) were listed. b Dual-luciferase reporter assay confirmed that NEU1 was the target of miR-23b-3p. c-d Relative NEU1 protein/GAPDH expressions after obesity-induced IR model construction and miR-23b-3p upregulation were measured with Western blot. GAPDH was employed as internal control. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). ^†††P < 0.001, vs. Control; ^***P < 0.001, vs. Control; ^{^^^}P < 0.001, vs. Model; ^###P < 0.001, vs. Acacetin+MC. WT: wild-type; MUT: mutated; M: mimic; NEU1: Neuraminidase 1

Fig. 3

Overexpressed NEU1 reversed the effects of miR-23b-3p upregulation on Treg and Th17 cell percentage. a-d Percentages of Treg cells (CD4⁺ CD25⁺ Foxp3⁺) and Th17 cells (CD4⁺ IL-17⁺) after obesity-induced IR model construction and injection of lentivirus carriers for miR-23b-3p mimic and NEU1 overexpression plasmid were calculated by flow cytometry. e-f Protein/GAPDH expressions of TGF-β1, IL-10, IL-17 and IL-6 after obesity-induced IR model construction and injection of lentivirus carriers for miR-23b-3p mimic and NEU1 overexpression plasmid were measured with Western blot. GAPDH was employed as internal control. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). ^***P < 0.001, vs. Control; ^{^^^}P < 0.001, vs. MC; ^##P < 0.01, ^###P < 0.001, vs. negative control (NC); ^ΔΔP < 0.01, ^ΔΔΔP < 0.001, vs. mimic (M); ^ξξξP < 0.001, vs. NEU1. Treg cells: regulatory T cells; Th17 cells: T helper 17 cells; TGF-β1: Transforming growth factor-β1

Fig. 4

Overexpressed NEU1 reversed the effects of upregulated miR-23b-3p in obesity-induced IR of mice. a Body weight of mice after obesity-induced IR model construction and injection of lentivirus carriers for miR-23b-3p mimic and NEU1 overexpression plasmid was measured. b Fat percent of mice after obesity-induced IR model construction and injection of lentivirus carriers for miR-23b-3p mimic and NEU1 overexpression plasmid was calculated. c-d Levels of fasting blood glucose (c) and fasting insulin (d) after obesity-induced IR model construction and injection of lentivirus carriers for miR-23b-3p mimic and NEU1 overexpression plasmid were determined. e-f AUC of mice for OGTT (e) and ITT (f) after obesity-induced IR model construction and injection of lentivirus carriers for miR-23b-3p mimic and NEU1 overexpression plasmid was quantified. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, vs. Control; ^{^}P < 0.05, ^{^^}P < 0.01, ^{^^^}P < 0.001, vs. MC; ^##P < 0.01, ^###P < 0.001, vs. NC; ^ΔP < 0.05, ^ΔΔP < 0.01, ^ΔΔΔP < 0.001, vs. M; ^ξξP < 0.01, ^ξξξP < 0.001, vs. NEU1

Fig. 5

Overexpressed NEU1 reversed the effects of upregulated miR-23b-3p on inflammatory cytokines levels and NEU1 expression in obese mice. a-d Levels of inflammatory cytokines CRP (a), IL-6 (b), TNF-α (c) and MCP1 (d) after obesity-induced IR model construction and injection of lentivirus carriers for miR-23b-3p mimic and NEU1 overexpression plasmid were detected with ELISA. e-f NEU1 protein/GAPDH expressions after obesity-induced IR model construction and injection of lentivirus carriers for miR-23b-3p mimic and NEU1 overexpression plasmid were measured with Western blot. GAPDH was employed as internal control. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). ^**P < 0.01, ^***P < 0.001, vs. Control; ^{^}P < 0.05, ^{^^}P < 0.01, ^{^^^}P < 0.001, vs. MC; ^##P < 0.01, ^###P < 0.001, vs. NC; ^ΔP < 0.05, ^ΔΔP < 0.01, ^ΔΔΔP < 0.001, vs. M; ^ξξP < 0.01, ^ξξξP < 0.001, vs. NEU1