Abstract
Acute kidney injury (AKI) is a common critical illness that involves multiple systems and multiple organs with a rapid decline in kidney function over short period. It has a high mortality rate and presents a great treatment challenge for physicians. Oleuropein, the main active constituent of Ilex pubescens Hook. et Arn. var. kwangsiensis Hand.-Mazz. displays significant anti-inflammatory activity, although oleuropein’s therapeutic effect and mechanism of action in AKI remain to be elucidated. The present study aimed to further clarify the mechanism by which oleuropein exerts effects on inflammation in vitro and in vivo. In vitro, the inflammatory effect and mechanism were investigated through ELISA, Western blotting, the thermal shift assay, co-immunoprecipitation, and immunofluorescence staining. Lipopolysaccharide (LPS) induced acute kidney injury was employed in an animal model to investigate oleuropein’s therapeutic effect on AKI and mechanism in vivo. The underlying mechanisms were investigated by Western blot analysis of kidney tissue. In LPS-stimulated macrophages, our data demonstrated that oleuropein significantly reduced the expression of inflammatory mediators like NO, IL-6, TNF-α, iNOS, and COX-2. Moreover, oleuropein inhibited NF-κB/p65 translocation, and had a negative regulatory effect on key proteins in the NF-κB and MAPK pathways. In addition, the thermal shift and co-immunoprecipitation assays revealed that oleuropein played an essential role in binding to the active sites of TLR4, as well as inhibiting TLR4 dimerization and suppressing the binding of TLR4 to MyD88. Oleuropein markedly alleviated LPS induced acute kidney injury, decreased serum creatinine and blood urea nitrogen (BUN) levels and proinflammatory cytokines. More importantly, the TLR4-MyD88-NF-κB/MAPK pathways were confirmed to play an important role in the oleuropein treatment of AKI. In this study, oleuropein exhibited excellent anti-inflammatory effects by regulating TLR4-MyD88-NF-κB/MAPK axis in vitro and in vivo, suggesting oleuropein as a candidate molecule for treating AKI.
Highlights
Acute kidney injury (AKI) is a clinical syndrome caused by a variety of etiologies and pathological mechanisms (Ronco et al, 2019; Jentzer et al, 2020)
TLR4 interacts with myeloid differentiation primary response gene 88 (MyD88) and transmit signals to the cells, which promotes the activation of transforming growth factor-beta-activated kinase 1 (TAK1), leading to the phosphorylation of mitogen-activated protein kinase (MAPK) and the IKK complex, which causes the signal transduction of MAPK and the NF-κB pathway, and triggers the release of inflammatory cytokines, such as TNF-a, IL-1β, and IL-6. (Lu et al, 2017; Ahmad et al, 2019; Yuan et al, 2019)
The results showed that OP could reduce the phosphorylation of JNK1/2, ERK1/2 and p38 MAPK induced by LPS, but had no effect on total JNK1/2, ERK1/2 and p38 MAPK protein (Figure 3B) The Dual-Glo luciferase assay was used to evaluate the effects of OP on LPS-induced AP-1 activation in
Summary
Acute kidney injury (AKI) is a clinical syndrome caused by a variety of etiologies and pathological mechanisms (Ronco et al, 2019; Jentzer et al, 2020). The protein A/G magnetic bead kit (#88802), TurboFect transfection reagents (#R0531), and quantitative PCR (qPCR) kits were purchased from Thermo Fisher Scientific (Grand Island, NY, United States). The cells were pretreated with OP (10, 20, and 40 μM) for 1 h, treated with LPS (1 μg/ml) for 8 h and with DAF-FM (1 μM) for 1 h at 37°C. J774A.1 cells were pretreated with OP (10,20, and 40 μM) for 1 h, co-treated with OP and LPS (1 μg/ml) for 18 h. The treated cells were collected and total protein was extracted. J774A.1 cells cultured in 96-well plates overnight were transiently transfected with pAP-1-luc plasmids and pRL-TK plasmid according to the manufacturer’s instructions. In the positive control group, the mice were given dexamethasone after AKI induction. A p-value of < 0.05 indicated a significant difference
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