Stefin B alleviates the gouty arthritis in mice by inducing the M2 polarization of macrophages

Abstract

The present study aims to explore the therapeutic effect of Stefin B on gouty arthritis (GA) and the polarization of macrophages in mice. Stefin B-overexpressed or knockdown M0 macrophages were constructed. The GA model was established in mice by injecting 25 mg/mL MSU, followed by a single injecting of Stefin B-overexpressing adenovirus vector (GA model + Stefin B OE) or an empty vector (GA model + Stefin B OE NC). Stefin B was found lowly expressed in M1 macrophages. CD206 was markedly upregulated and IL-10 release was signally increased in Stefin B-overexpressed macrophages. In gouty arthritis mice, marked redness and swelling were observed in the ankle joint. Dramatical infiltration of inflammatory cells was observed in the GA model and GA model + Stefin B OE NC groups, which was suppressed in the Stefin B OE group. Increased proportion of F4/80⁺CD86⁺ cells observed in GA mice was markedly repressed by Stefin B overexpression, accompanied by the declined level of Caspase-1 and IL-17. Collectively, Stefin B alleviated the GA in mice by inducing the M2 polarization of macrophages.

Keywords: Acute gouty arthritis; IL-17; Macrophages polarization; Stefin B.

Fig. 1

Macrophage polarization from THP-1 cells was identified. A The mRNA level of TNF-α was detected by the RT-PCR assay. B The level of CD86 and CD206 was checked by flow cytometry (*p < 0.05 vs M0)

Fig. 2

Stefin B was differentially expressed in M1/M2 macrophages. The protein level of Stefin B in M0, M1, and M2 macrophages was determined by western blotting assay (*p < 0.05 vs M0)

Fig. 3

The identification of the overexpression and knockout of Stefin B in M0 macrophages. The level of Stefin B in pcDNA3.1-Stefin B-transfected M0 macrophages was checked by RT-PCR (A) and western blotting assay (B). The Stefin B expression in siRNAs-transfected M0 macrophages was checked by RT-PCR (C) and western blotting assay (D). (*p < 0.05 vs Control, #p < 0.05 vs Stefin B OE NC or si-Stefin B NC)

Fig. 4

The M2 polarization could be facilitated by Stefin B. A The level of IL-6 and IL-10 released by macrophages was checked by ELISA. B The percentage of CD206 + macrophages was determined by the flow cytometry assay (*p < 0.05 vs Control, #p < 0.05 vs Stefin B OE NC or si-Stefin B NC)

Fig. 5

The identification of Stefin B-overexpressed adenovirus vector. A The picture of the gel electrophoresis. B The mRNA level of Stefin B was checked by RT-PCR (*p < 0.05 vs Control, #p < 0.05 vs Stefin B OE NC)

Fig. 6

The successful establishment of GA model in mice. A The ankle joint picture of mice in the control and GA model groups. B The ankle circumference of the mice in the control and GA model groups was evaluated (*p < 0.05 vs Control)

Fig. 7

Stefin B overexpression alleviated the GA symptom in mice. The pathological state in the ankle joint tissue of mice was checked by HE staining assay

Fig. 8

Stefin B overexpression repressed the M1 polarization and facilitated the M2 polarization of macrophages in the ankle joint tissue of GA mice. A The expression of CD86 and F4/80 in macrophages was detected by the double immunofluorescence assay. B The expression of CD206 and F4/80 in macrophages was detected by the double immunofluorescence assay

Fig. 9

Stefin B overexpression suppressed the inflammation in GA mice. A The mRNA level in the ankle joint tissue was checked by RT-PCR assay. B The serum IL-17 level was detected by the ELISA assay (*p < 0.05 vs Control, #p < 0.05 vs GA model, &p < 0.05 vs GAmodel + Stefin B OE NC)