NADPH oxidase 4 regulate the glycolytic metabolic reprogramming of microglial cells to promote M1 polarization.
Liping ZhaiShuiliang RuanJin WangQiaobing GuanLi ZhaPublished in: Journal of biochemical and molecular toxicology (2023)
This work aimed to investigate the role and mechanism of NADPH oxidase 4 (NOX4) in the polarization of microglial cells. Microglial cells were transfected with the NOX4 overexpression plasmid (pGL3-NOX4), and later treated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to induce its M1 polarization. Later, the F4/80 + CD86 + cell proportion was detected by flow cytometry (FCM), the inflammatory factor expression levels were analyzed through enzyme-linked immunosorbent assay (ELISA), while ionized calcium binding adapter molecule 1 (IBA-1) and PKM2 expression were measured by immunofluorescence (IF) staining. In addition, dichlorodihydrofluorescein diacetate probe was utilized to detect the reactive oxygen species (ROS) levels, glucose uptake, and glycolysis, as well as lactic acid level. The expression of glycolytic enzymes PKM2, HK2, and citrate (Si)-synthas (CS) was detected by Western-blot (WB) assay. Moreover, the polarization level of microglial cells was detected after ROS expression was suppressed by the ROS inhibitor N-acetylcysteine (NAC). In mouse experiments, LPS was applied in inducing central neuroinflammation in NOX4 knockdown mouse model (KO) and wild-type mice (WT). Thereafter, the inflammatory factor levels and lactic acid level in mouse tissues were detected; IBA-1 and CD86 expression in mice was measured by IF staining; and the expression of glycolytic enzymes PKM2, HK2, and CS in the central nervous system (CNS) was also detected. After NOX4 overexpression in microglial cells, the M1 polarization level was upregulated, the F4/80 + CD86 + cell proportion increased, and inflammatory factors were upregulated. At the same time, the expression of glycolytic enzymes PKM2, HK2, and CS was upregulated. NAC pretreatment suppressed the effects of NOX4, reduced the F4/80 + CD86 + cell proportion, and suppressed the expression of PKM2, HK2, and CS. In the mouse model, the expression levels of CD86 in KO group decreased, and the inflammatory factors were also downregulated. NOX4 promotes glycolysis of microglial cells via ROS, thus accelerating M1 polarization and inflammatory factor expression. In this regard, NOX4 is promising as a new target for the treatment of neuroinflammation.
Keyphrases
- reactive oxygen species
- poor prognosis
- induced apoptosis
- inflammatory response
- cell cycle arrest
- lps induced
- mouse model
- lipopolysaccharide induced
- oxidative stress
- transcription factor
- binding protein
- cell death
- neuropathic pain
- traumatic brain injury
- dendritic cells
- cell proliferation
- long non coding rna
- escherichia coli
- lactic acid
- endoplasmic reticulum stress
- stem cells
- metabolic syndrome
- brain injury
- adipose tissue
- skeletal muscle
- cell therapy
- high throughput
- ionic liquid
- room temperature
- blood brain barrier
- crispr cas
- spinal cord
- anti inflammatory
- cerebrospinal fluid
- nk cells
- glycemic control