Yuanhuacine modulates lipopolysaccharide-induced interleukin-6 through regulation of the JAK1/STAT3 pathway and prevents tubular damage in acute kidney injury

Abstract

Inflammation is an immune response that activates immune cells to protect the host from infection or tissue damage; however, excessive inflammation can lead to sepsis and acute kidney injury (AKI). Yuanhuacine (YC), a physiologically active compound derived from Daphne genkwa flowers, has demonstrated its therapeutic potential in various diseases, including inflammatory diseases and cancer. However, the underlying molecular mechanisms by which YC regulates inflammatory cytokines and exerts efficacy against AKI remain to be elucidated. The present study aimed to investigate the role of YC in regulating cytokines in human macrophages and to evaluate its protective effect in a mouse model of AKI. Lipopolysaccharide (LPS) was used to stimulate THP-1 macrophages in vitro, and LPS was administered intraperitoneally to establish an in vivo AKI model. LPS treatment significantly increased interleukin 6 (IL-6) expression in both macrophages and in mice with AKI. However, YC treatment effectively reduced IL-6 production by inhibiting the activation of Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) in macrophages, and YC was confirmed to inhibit LPS-induced tubular damage in the mouse model of AKI. In conclusion, YC may serve as a potential therapeutic agent in the prevention of AKI and other IL-6-related inflammatory diseases by promoting JAK1/STAT3 dephosphorylation to facilitate inflammation resolution.

Keywords: AKI; IL-6; JAK1/STAT3 pathway; LPS; YC.

Figure 1

Structure of YC and its effect on inflammatory cytokines in differentiated THP-1 cells. (A) Chemical structure of YC. (B) Differentiated THP-1 cells were treated with various concentrations of YC, with or without LPS for 24 h. (C) Time-course analysis of inflammatory cytokine mRNA levels (0-8 h). (D) mRNA levels of LPS-induced inflammatory cytokines after 4 h treatment with various doses of YC. (E) Time-course analysis of IL-6 protein levels (0-24 h). (F) IL-6 protein levels after treatment with various concentrations of YC. Cell viability was assessed using the Cell Counting Kit-8 assay. The mRNA and protein levels of cytokines were analyzed using reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. LPS (5 ng/ml) was used in all experiments. Data are presented as the mean ± standard deviation from three independent experiments. Statistical significance was determined using one-way and two-way analysis of variance. ^###P<0.001 vs. control; ^$$$P<0.001, ^$P<0.05 vs. LPS; ns, not significant; YC, yuanhuacine; LPS, lipopolysaccharide; IL, interleukin.

Figure 2

YC inhibits LPS-induced phosphorylation of JAK1 and STAT3 in THP-1 cells. (A) Differentiated THP-1 cells were treated with 5 ng/ml LPS and 200 nM YC for up to 12 h. The activation of kinases as well as transcription factors was evaluated by western blotting analysis following LPS stimulation. (B) Quantification of phosphorylated JAK1 and STAT3 proteins after treatment with increasing concentrations of YC. White lines indicate separate groups. Phosphorylation levels were normalized to total JAK1 and STAT3 expression and quantified using ImageJ software. Data are presented as the mean ± standard deviation from three independent experiments. Statistical significance was determined using one-way analysis of variance. ^###P<0.001, ^##P<0.01 vs. control; ^$$P<0.01 vs. LPS; ns, not significant; YC, yuanhuacine; LPS, lipopolysaccharide; JAK1, Janus kinase 1; STAT3, signal transducer and activator of transcription 3; p-, phosphorylated.

Figure 3

YC attenuates LPS-induced cytoplasmic-to-nuclear translocation of phosphorylated STAT3 in THP-1 cells. THP-1 cells were treated with 5 ng/ml LPS for 2 h in the absence or presence of YC at three concentrations: 8, 40, and 200 nM. The subcellular localization of STAT3 was assessed by multiple methods. (A) Western blot analysis of cytoplasmic and nuclear fractions was performed to determine the localization of STAT3 protein. (B) Confocal microscopy image showing the intracellular distribution of p-STAT3. (C) Confocal microscopy image showing the intracellular distribution of total STAT3. β-Actin was used as a cytoplasmic marker, and Lamin B served as a nuclear marker. Nuclei were stained with DAPI (blue), and STAT3 (total and p-STAT3) was visualized in green. Scale bars: 10 µm. YC, yuanhuacine; LPS, lipopolysaccharide; STAT3, signal transducer and activator of transcription 3; p-, phosphorylated.

Figure 4

Effect of YC on IL-6 expression compared with Janus kinase 1 (filgotinib) and signal transducer and activator of transcription 3 (S3I-201) inhibitors. THP-1 cells were treated with 5 ng/ml LPS for 24 h in the presence of varying concentrations of (A) filgotinib, (B) S3I-201 and (C) YC. IL-6 levels were measured in the supernatants of the treated cells using enzyme-linked immunosorbent assay. Data are presented as the mean ± standard deviation from three independent experiments. Statistical differences were analyzed using one-way analysis of variance. ^###P<0.001, ^##P<0.01 vs. control; ^$$$P<0.001, ^$$P<0.01 vs. LPS; ns, not significant; YC, yuanhuacine; LPS, lipopolysaccharide; IL, interleukin.

Figure 5

Protective effects of YC in an LPS-induced AKI mouse model. Renal dysfunction was evaluated by measuring (A) BUN and (B) sCr levels, and (C) tubular injury was assessed by scoring histological damage (n=5). (D) A representative image of H&E staining and immunohistochemistry images in kidney tissue. The arrows indicate tubular degeneration. In kidney tissues of mice with AKI (n=5), the expression levels of (E) KIM-1, (F) NGAL and (G) IL-6, markers of renal injury, were analyzed by quantitative polymerase chain reaction. (H) Western blot analysis of JAK1 and STAT3 activation in kidney tissues of AKI mice (n=4) following YC treatment. The image density was analyzed using ImageJ. Scale bars: 500 µm (40x), 100 µm (100x, 200x), and 50 µm (400x). Data are presented as the mean ± standard deviation. Statistical significance was determined using one-way analysis of variance. ^###P<0.001, ^##P<0.01, ^#P<0.05 vs. control; ^$$$P<0.001, ^$$P<0.01, ^$P<0.05 vs. LPS; ns, not significant; AKI, acute kidney injury; H&E, hematoxylin and eosin; KIM-1, kidney injury molecule 1; NGAL, neutrophil gelatinase-associated lipocalin; BUN, blood urea nitrogen; sCr, serum creatinine; YC, yuanhuacine; LPS, lipopolysaccharide; IL-6, interleukin-6; JAK1, Janus kinase 1; STAT3, signal transducer and activator of transcription 3.