Kinsenoside screening with a microfluidic chip attenuates gouty arthritis through inactivating NF-κB signaling in macrophages and protecting endothelial cells

Abstract

Gouty arthritis is a rheumatic disease that is characterized by the deposition of monosodium urate (MSU) in synovial joints cause by the increased serum hyperuricemia. This study used a three-dimensional (3D) flowing microfluidic chip to screen the effective candidate against MSU-stimulated human umbilical vein endothelial cell (HUVEC) damage, and found kinsenoside (Kin) to be the leading active component of Anoectochilus roxburghi, one of the Chinese medicinal plant widely used in the treatment of gouty arthritis clinically. Cell viability and apoptosis of HUVECs were evaluated, indicating that direct Kin stimulation and conditioned medium (CM) from Kin-treated macrophages both negatively modulated with MSU crystals. Additionally, Kin was capable of attenuating MSU-induced activation of nuclear factor-κB/mitogen-activated protein kinase (NF-κB/MAPK) signaling, targeting IκB kinase-α (IKKα) and IKKβ kinases of macrophages and influencing the expressions of NF-κB downstream cytokines and subsequent HUVEC bioactivity. Inflammasome NLR pyrin domain-containing 3 (NALP3) and toll-like receptor 2 (TLR2) were also inhibited after Kin treatment. Also, Kin downregulated CD14-mediated MSU crystals uptake in macrophages. In vivo study with MSU-injected ankle joints further revealed the significant suppression of inflammatory infiltration and endothelia impairment coupled with alleviation of ankle swelling and nociceptive response via Kin treatments. Taken together, these data implicated that Kin was the most effective candidate from Anoectochilus roxburghi to treat gouty arthritis clinically.

Figure 1

Schematic graph of the integrated microfluidic 3D flowing GCC for screening. (A) The vivo-like system consists of medium inputs, parallel CGCs and CCCs. (Ba) Mixture of blue colorant with red colorant in CGC zone displayed a well-proportioned concentration distribution. (b) Mixture of crystal violet and PBS demonstrated gradient color depth, which reflect varying drug concentrations. (c) The indicated distribution of drug concentrations in the CCC zone. (C) The complete view of GCCs. The data are presented as the means±S.D. All data were obtained from at least three independent experiments

Figure 2

Microfluidic screening of effective candidate from A. roxburghi. (A) The apoptotic HUVECs under 3D flowing condition in response to various concentrations of MSU crystals were compared (magnification, × 100). (B) The apoptotic HUVECs under 3D flowing condition pre-treated with 300 μg/ml MSU crystals and stimulated with varying concentrations of Sti (a), Ua (b), Gas (c), Kae (d) and Kin (e) were prepared (magnification, × 100). (C) The quantitative cell viability of HUVECs under 3D flowing conditions in response to various concentrations of MSU crystals were compared. (d) The quantitative cell viability of HUVECs under 3D flowing conditions pre-treated with 300 μg/ml MSU crystals and stimulated with varying concentrations of Sti (a), Ua (b), Gas (c), Kae (d) and Kin (e) were prepared. The data are presented as the means±S.D. **P<0.05 compared with control. All data were obtained from at least three independent experiments

Figure 3

Effects of Kin on MSU-stimulated HUVECs. (A) The chemical structural formula of Kin. (Ba) Cell viability were measured in Kin-treated HUVECs. (b) Cell viability were measured in HUVECs pre-treated with MSU crystals and stimulated with various concentrations of Kin. (C) Apoptosis rates were analyzed with HUVECs pre-treated with MSU crystals and stimulated with various concentrations of Kin. (D) Expressions of apoptosis-related proteins (Bax, Bcl-2, cleaved caspase-3, caspase-3) were compared in HUVECs pre-treated with MSU crystals and stimulated with various concentrations of Kin. The data are presented as the means±S.D. **P<0.05 compared with 0 μg/ml of Kin. ^#P<0.05 compared with control. All data were obtained from at least three independent experiments

Figure 4

Effects of Kin-treated MSU-CM from macrophages on HUVECs. (a) Cell viability were measured in HUVECs treated with CM from both MSU crystals and Kin-treated macrophage cells. (b) Apoptosis rates were analyzed with HUVECs treated with CM from both MSU crystals and Kin-treated macrophages. (c) Expressions of apoptosis-related proteins (Bax, Bcl-2, cleaved caspase-3, caspase-3) were compared in HUVECs treated with CM from MSU crystals and Kin-treated macrophages. The data are presented as the means±S.D. **P<0.05 compared with 0 μg/ml of Kin. ^#P<0.05 compared with control. All data were obtained from at least three independent experiments

Figure 5

Effects of Kin on MSU-induced NF-κB/MAPK activation. (a) Transfected RAW264.7 macrophage cells were incubated with Kin and MSU for 7 h. The luciferase activity for NF-κB was evaluated and normalized to the control. (b) Expression of NF-κB signaling-associated upstream proteins (TRAF-6, p-TAK-1/TAK-1, p-IKKαβ/IKKαβ) were compared in macrophages treated with MSU crystals and Kin. (c) Expression of NF-κB signaling-associated downstream proteins (p-IκBα/IκBα, p-p65/p65) was compared in macrophages treated with MSU crystals and Kin. (d) Molecular docking of IKKα and IKKβ with Kin. (e) Expressions of MAPK signaling-associated proteins (p-JNK/JNK, p-Erk/Erk, p38/p-p38) were compared in macrophages treated with MSU crystals and Kin. The data are presented as the means±S.D. **P<0.05 compared with 0 μg/ml of Kin. ^#P<0.05 compared with control. All data were obtained from at least three independent experiments

Figure 6

Effects of Kin on the expression of MSU-induced NF-κB downstream inflammatory cytokines and inflammasome and schematic diagram of the proposed mechanisms of antigouty arthritis through modulation of macrophages. (A) mRNA expressions of NF-κB downstream IL-1β, IL-6, TNF-α, NO and PGE₂ in macrophages treated with MSU crystals and Kin. (B) Measurement of the secretion of NF-κB downstream IL-1β, IL-6, TNF-α, NO and PGE₂ in macrophages treated with MSU crystals and Kin. (C) Expressions of NALP3 and TLR2 were compared in macrophages treated with MSU crystals and Kin. (D) Effects of Kin on MSU-stimulated expression of COX-1 in HUVECs and COX-2 in inflammatory macrophages. (E) Kin inactivates NF-κB pathway via targeting of IKK kinases, abrogating phosphorylation of IκBα and p65, downregulating expression of IL-1β, IL-6, TNF-α, NO and PGE₂ in macrophages, attenuating MSU-mediated HUVECs impairment. The data are presented as the means±S.D. **P<0.05 compared with 0 μg/ml of Kin. ^#P<0.05 compared with control. All data were obtained from at least three independent experiments

Figure 7

Determination of CD14-mediated MSU crystals uptake in macrophages after Kin. (a) Expressions of CD14 were compared in macrophages treated with MSU crystals and Kin. (b) Uptake of MSU crystals was analyzed with flow cytometry as an increase in the macrophage side-scatter high (SSC-H). The data are presented as the means±S.D. **P<0.05 compared with 0 μg/ml of Kin. ^#P<0.05 compared with control. All data were obtained from at least three independent experiments

Figure 8

In vivo examination of anti-inflammatory effects of Kin against gouty arthritis. (A) Histological analyses of rat ankle joints injected with MSU crystals and corresponding drugs (a, magnification, × 100, × 200; b, magnification, × 400). Black arrows indicate MSU-affected blood vessels. (B) Histological analyses of stomach (a) and kidney (b) after rats received injection of MSU crystals and corresponding drugs (magnification, × 200). (C) Perimeter assessments of ankles of rats injected with MSU crystals and corresponding drugs. (D) The effects of Kin on nociceptive reactions caused by hot water (tail-flick response, a) and acetic acid (writhing response, b). The data are presented as the means±S.D. **P<0.05 compared with 0 μg/ml of Kin. ^#P<0.05 compared with control. All data were obtained from at least three independent experiments