Immortalized Mouse Microglia (SIM-A9)

Alterations in RTP4 Expression Following LPS Treatment in SIM-A9 Microglial Cell Line

Receptor transporter protein 4 (RTP4), known for its role as a receptor chaperone for class A GPCRs, has emerged as an important player in inflammatory regulation, especially in peripheral immune responses. As microglia are key players in neuroinflammation, understanding RTP4's role is vital. By examining RTP4 and inflammation-related gene expression changes in response to LPS, Fujita et al. aims to uncover RTP4's function as an inflammation-responsive molecule in microglia.

Quantitative PCR reveals that 24 hours of LPS treatment significantly elevates RTP4 mRNA levels in a concentration-dependent manner (Fig. 1A). RTP4 upregulation is robust from 3 to 24 hours post-treatment (Fig. 1B). LPS does not significantly affect other RTPs but slightly increases RTP3, specifically upregulating RTP4 (Fig. 1C). Immunofluorescence analysis shows enhanced RTP4 expression in SIM-A9 microglial cells treated with LPS for 6, 12, and 24 hours, but not at 48 hours (Fig. 2, upper panel). Data quantification confirms increased RTP4 immunoreactivity at these times (Fig. 2, lower panel). Thus, LPS temporarily elevates RTP4 mRNA and protein levels (Fig. 2).

Fig. 1. Changes in RTP4 mRNA levels after LPS stimulation in SIM-A9 microglial cell line (Fujita W and Kuroiwa Y, 2024).

Fig. 2. The effect of LPS stimulation on the expression levels of RTP4 determined by immunofluorescent analysis (Fujita W and Kuroiwa Y, 2024).

Impacts of HJE and luteolin on pro-inflammatory cytokines and pro-inflammatory intermediaries in SIM-A9 microglia

Humulus japonicus (HJ) possesses anti-inflammatory and antioxidant properties which make it a traditional remedy for skin and respiratory conditions while also demonstrating possible neuroprotective benefits. Wang et al. investigates how HJ ethanol extract (HJE) and its component luteolin reduce inflammation in microglial cells (SIM-A9) activated by LPS.

Microglia defend the CNS through their activation of immune responses. Abnormal activation of these cells leads to the production of pro-inflammatory intermediaries (ROS, COX2, PGE2, NO, iNOS) and cytokines (IL-1β, TNF-α, IL-6) which results in neurodegenerative diseases like Parkinson's and cognitive decline. Controlling neuroinflammation is vital for treatment. Microglial activation through LPS stimulation results in the production of neurotoxic intermediaries which lead to neuron death. Our research demonstrates that LPS by itself elevates NO, iNOS, PGE2, COX2 levels (Fig. 3), and cytokine production (IL-1β, TNF-α, IL-6) (Fig. 4). When cells receive pretreatment with HJE and luteolin before receiving LPS exposure, they show limited increases in levels of inflammatory cytokines and intermediaries. SIM-A9 microglial cells treated with LPS show reduced inflammation when pretreated with HJE and luteolin.

Fig. 3. The effects of HJE and luteolin NO production (A), iNOS expression levels (B), PGE2 generation (C), and COX2 expression degrees (D) in LPS-stimulated microglia cells SIM-A9 (Wang F, Cho B O, et al., 2022).

Fig. 4. Impacts of HJE and luteolin on IL-1β(A), TNF-α(B) along with IL-6 (C) production in the microglial cells stimulated by LPS (Wang F, Cho B O, et al., 2022).