Highly electronegative low‐density lipoprotein L5 evokes microglial activation and creates a neuroinflammatory stress via Toll‐like receptor 4 signaling

Abstract

Atherogenic risk factors, such as hypercholesterolemia, are associated with increased risk of neurodegeneration, especially Alzheimer's dementia. Human plasma electronegative low-density lipoprotein [LDL(-)], especially L5, may serve as an important contributing factor. L5 promoting an inflammatory action in atherosclerosis has been extensively studied. However, the role of L5 in inducing neuroinflammation remains unknown. Here, we examined the impact of L5 on immune activation and cell viability in cultured BV-2 microglia. BV-2 cells treated with lipopolysaccharide or human LDLs (L1, L5, or oxLDL) were subjected to molecular/biochemical assays for measuring microglial activation, levels of inflammatory factors, and cell survival. A transwell BV-2/N2a co-culture was used to assess N2a cell viability following BV-2 cell exposure to L5. We found that L5 enables the activation of microglia and elicits an inflammatory response, as evidenced by increased oxygen/nitrogen free radicals (nitric oxide, reactive oxygen species, and peroxides), elevated tumor necrosis factor-α levels, decreased basal interleukin-10 levels, and augmented production of pro-inflammatory proteins (inducible nitric oxide synthase and cyclooxygenase-2). L5 also triggered BV-2 cell death primarily via apoptosis. These effects were markedly disrupted by the application of signaling pathway inhibitors, thus demonstrating that L5 interacts with Toll-like receptor 4 to modulate multiple pathways, including MAPKs, PI3K/Akt, and NF-κB. Decreased N2a cell viability in a transwell co-culture was mainly ascribed to L5-induced BV-2 cell activation. Together, our data suggest that L5 creates a neuroinflammatory stress via microglial Toll-like receptor 4, thereby leading to the death of BV-2 microglia and coexistent N2a cells. Atherogenic L5 possibly contributes to neuroinflammation-related neurodegeneration.

A Novel Quinolyl‐Substituted Analogue of Resveratrol Inhibits LPS‐Induced Inflammatory Responses in Microglial Cells by Blocking the NF‐κB/MAPK Signaling Pathways

“…It has been reported that TLR4 is responsible for microglia‐mediated neuroinflammation . The extracellular domain of TLR4 is able to recognize and bind with LPS, which triggers intracellular signaling events that can activate the MAPK and NF‐κB signaling pathways . This study showed that RV01 potently suppressed LPS‐induced TLR4 protein expression.…”

“…RV01 decreased TLR4 protein expression, inhibited NADPH oxidase activation, attenuated ROS production, suppressed the corresponding downstream MAPKs and NF‐κB, and subsequently reduced the expression of pro‐inflammatory mediators in LPS‐activated microglia. As mentioned above, TLR4 extracellular domain is able to recognize and bind with LPS, which sequentially triggers the intracellular signaling complex that can activate downstream signaling pathways, including MAPK and NF‐κB signaling pathways . Therefore, it can be speculated that RV01 might exert anti‐inflammatory effect through regulating TLR4, which may serve as the primary target of RV01.…”

Acute glucose fluctuation impacts microglial activity, leading to inflammatory activation or self-degradation

“…Activation is essential for a cell’s response to external stimuli and is typically featured by protein neo-synthesis or elevated enzyme activity to accelerate and amplify intracellular biochemical reactions. Given that iNOS and COX-2 upregulation correlates with pathological stress, such as neuroinflammation characterized by microglial activation and the production of inflammatory factors 17 . The neo-synthesis of these inflammatory proteins was assayed as a marker of microglial activation.…”

Electronegative Low-Density Lipoprotein L5 Impairs Viability and NGF-Induced Neuronal Differentiation of PC12 Cells via LOX-1

“…The yielding LDL at a density of 1.019 to 1.063 g/mL was treated with 5 mM EDTA and nitrogen to avoid ex vivo oxidation. After removing salts by dialysis (20 mM Tris-HCl [pH 8.0], 0.5 mM EDTA and 0.01% NaN 3 ), LDL was further separated into subfractions of L1 to L5 according to electrical charge by using a fast protein liquid chromatography with a UnoQ12 column (Bio-Rad Laboratories, Inc., Hercules, CA, USA), as described previously [ 18 , 59 ]. OxLDL was prepared by incubating L5-free LDL (L1) with 5 μM CuSO 4 at 37 °C for 24 h. All LDL particles were sterilized by passing through 0.22 μm filters before using for cell experiments.…”

“…Cells were collected and homogenized in ice-cold RIPA buffer as described previously [ 59 ]. After a 20-min incubation at 4 °C, cell extracts were centrifuged at 14,000× g for 10 min and protein concentration in the supernatant was determined with a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA).…”