Kaempferol alleviates adipose tissue inflammation and insulin resistance in db/db mice by inhibiting the STING/NLRP3 signaling pathway

Abstract

Chronic inflammation induced by obesity plays a crucial role in the pathogenesis of insulin resistance. The infiltration of macrophages into adipose tissues contributes to adipose tissue inflammation and insulin resistance. Kaempferol, a flavonoid present in various vegetables and fruits, has been shown to possess remarkable anti-inflammatory properties. In this study, we used leptin receptor-deficient obese mice (db/db) as an insulin-resistant model and investigated the effects of kaempferol treatment on obesity-induced insulin resistance. Our findings revealed that the administration of kaempferol (50 mg/kg/day, for 6 weeks) significantly reduced body weight, fat mass, and adipocyte size. Moreover, it effectively ameliorated abnormal glucose tolerance and insulin resistance in db/db mice. In the adipose tissue of obese mice treated with kaempferol, we observed a reduction in macrophage infiltration and a downregulation of mRNA expression of M1 marker genes TNF-α and IL-1β, accompanied by an upregulation of Arg1 and IL-10 mRNA expression. Additionally, kaempferol treatment significantly inhibited the STING/NLRP3 signaling pathway in adipose tissue. In vitro experiments, we further discovered that kaempferol treatment suppressed LPS-induced inflammation through the activation of NLRP3/caspase 1 signaling in RAW 264.7 macrophages. Our results suggest that kaempferol may effectively alleviate inflammation and insulin resistance in the adipose tissue of db/db mice by modulating the STING/NLRP3 signaling pathway.

Keywords: STING/NLRP3; adipose tissue inflammation; db/db mice; insulin resistance; kaempferol; obesity.

Figure 1

Chronic administration of kaempferol demonstrates a pronounced effect on improving insulin sensitivity in db/db mice. (A) body weight, (B) glucose tolerance test (GTT), and (C) insulin tolerance test (ITT). Data are presented as mean ± s.e.m. (n = 7−8), ***P < 0.001 vs db/dm, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs db/db.

Figure 2

Kaempferol treatment improves HFD-induced obesity and fat accumulation in mice. (A) Representative images of epididymal adipose tissue from the mice. (B) Weights of eWAT from the mice. (C) Representative images of subcutaneous adipose tissue from the mice. (D) Weights of sWAT from the mice. H&E staining of eWAT (E). Scale bar, 100 mm. (F) Quantitative analyses of adipocyte size. Data are presented as mean ± s.e.m. (n = 4–5), ***P < 0.001 vs db/dm, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs db/db.

Figure 3

Kaempferol alleviates inflammation in epididymal adipose tissue of db/db mice. (A) F4/80 immunohistochemical analysis of eWAT; scale bar = 100 μm (n = 4–5). (B) The ratio of all F4/80-positive pixels was counted in each fraction of eWAT (n = 4–5). (C, D) The expression of macrophage and M1 pro-inflammatory genes in eWAT was quantified by RT-PCR (n = 4–5). (E, F) The relative mRNA levels of M2 anti-inflammatory genes in eWAT of mice. The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 vs db/dm, ^#P < 0.05, ^##P < 0.01 vs db/db.

Figure 4

Effects of kaempferol on the STING/NLRP3-caspase1 pathway in adipose tissue. (A) STING immunohistochemical analysis of eWAT; scale bar = 20 μm (n = 4–5). (B) The ratio of all STING-positive pixels was counted in each fraction of eWAT (n = 6–8). (C) Western blot analysis of NLRP3, caspase 1, and IL-1β (n = 4–5). (D–G) Relative densitometric values. The data are presented as the means ± s.e.m. **P < 0.01, ***P < 0.001 vs db/dm, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs db/db.

Figure 5

Kaempferol represses LPS-induced inflammatory cytokine secretion and NLRP3 inflammasome in RAW 264.7 macrophages. (A) Viability of cells after kaempferol treatment determined by the CCK-8 method. (B) Inflammatory cytokine mRNA expression levels quantified by RT-qPCR. (C, D) The protein and mRNA expressions of NLRP3, caspase 1, and IL-1β were measured by western blot and RT-qPCR analysis, respectively. Data were collected from three independent experiments and expressed as the mean ± s.e.m. *P < 0.05,**P < 0.01,***P < 0.001 vs control, ^##P < 0.01, ^###P < 0.001 vs LPS group.