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Updated: Jul 15, 2025
Non-thermal Infrared Light Treatment of Ischemia/Reperfusion Injury and Subsequent Analysis of Macrophage Differentiation
Published on: December 30, 2021
Roflumilast attenuates neuroinflammation post retinal ischemia/reperfusion injury by regulating microglia phenotype
Yuyan Guo1, Chunlian Ou2, Naiyuan Zhang3
1Department of Ophthalmology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China; Department of Ophthalmology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, Guangdong, China.
Purpose:
The abnormal polarisation of microglial cells (MGs) following retinal ischemia/reperfusion (RIR) initiates neuroinflammation and progressive death of retinal ganglion cells (RGCs), causing increasingly severe and irreversible visual dysfunction. Roflumilast (Roflu) is a promising candidate for treating neuroinflammatory diseases. This study aimed to explore whether Roflu displayed a cytoprotective effect against RIR-induced neuroinflammation and to characterise the underlying signalling pathway.
Methods:
The effects and mechanism of Roflu against RIR injury were investigated in C57BL/6J mice and the BV2 cell line. We used quantitative real-time PCR and enzyme-linked immunosorbent assay to examine the levels of inflammatory factors. Furthermore, haematoxylin and eosin and immunofluorescence (IF) stainings were used to assess the morphology of the retina and the states of MGs and RGCs. Reactive oxygen species (ROS) levels were examined using a ROS assay kit, while whole-genome sequencing analysis was conducted to identify altered pathways and molecules. Western blotting and IF staining were used to quantify the proteins associated with the nuclear factor erythroid 2-related factor 2 (Nrf2)/stimulator of interferon gene (STING)/nuclear factor kappa beta (NF-κB) pathway.
Results:
MG polarisation includes the pro-inflammatory and neurotoxic M1 phenotype as well as the anti-inflammatory and neuroprotective M2 phenotype. Roflu significantly attenuated MG activation and contributed to a shift in the MG phenotype from M1 to M2. Moreover, Roflu decreased ROS release and increased heme oxygenase 1 and NAD(P)H quinone oxidoreductase 1 expression. In vitro and in vivo experiments validated that Roflu exerted its neuroprotective effects primarily by upregulating the Nrf2/STING/NF-κB pathway. However, these effects were abrogated when the Nrf2 expression was inhibited by pharmacological or genetic manipulation.
Conclusions:
Roflu suppressed RIR-induced neuroinflammation by driving the shift of MG polarisation from M1 to M2 phenotype, which was mediated by the upregulation of the Nrf2/STING/NK-κB pathway.
Insights
Roflumilast reduces neuroinflammation and protects retinal cells after injury by shifting microglial polarization. This cytoprotective effect is mediated by the Nrf2/STING/NF-κB pathway, offering a potential treatment for visual dysfunction.
Area of Science:
- Neuroscience
- Ophthalmology
- Immunology
Background:
- Retinal ischemia/reperfusion (RIR) causes neuroinflammation and retinal ganglion cell (RGC) death, leading to irreversible vision loss.
- Abnormal microglial cell (MG) polarization contributes to RIR-induced neuroinflammation.
- Roflumilast (Roflu) is investigated for its potential therapeutic effects in neuroinflammatory conditions.
Purpose of the Study:
- To investigate the cytoprotective effects of Roflumilast against RIR-induced neuroinflammation.
- To elucidate the underlying signaling pathway involved in Roflumilast's protective mechanism.
Main Methods:
- Experiments were conducted in C57BL/6J mice and BV2 cell lines.
- Quantitative real-time PCR and ELISA measured inflammatory factors.
- Histological and immunofluorescence staining assessed retinal morphology, MG, and RGC states.
- Western blotting and whole-genome sequencing analyzed the Nrf2/STING/NF-κB pathway and identified altered molecules.
Main Results:
- Roflumilast significantly attenuated MG activation and shifted their phenotype from pro-inflammatory M1 to anti-inflammatory M2.
- Roflu decreased reactive oxygen species (ROS) levels and increased antioxidant enzyme expression (heme oxygenase 1, NAD(P)H quinone oxidoreductase 1).
- Neuroprotection was primarily mediated by upregulating the Nrf2/STING/NF-κB pathway, with effects abrogated by Nrf2 inhibition.
Conclusions:
- Roflumilast suppresses RIR-induced neuroinflammation by promoting M2 MG polarization.
- The mechanism involves the upregulation of the Nrf2/STING/NF-κB pathway.
- Roflumilast demonstrates therapeutic potential for RIR-induced visual dysfunction.
