Effect and mechanism of VSIG4 gene mutation on the function of microglia in retinitis pigmentosa

Authors: Xu Chunlong, Zhang Guowei, Du Jun, Jia Zhen, Wang Jingping, Wang Ziwen, Li Yang
Lu Hong
DOI: 10.3760/cma.j.cn115989-20240304-00063
   

Citation:

Xu Chunlong, Zhang Guowei, Du Jun, et al. Effect and mechanism of VSIG4 gene mutation on the function of microglia in retinitis pigmentosa[J]. Chin J Exp Ophthalmol, 2024, 42(10): 898-908. DOI: 10.3760/cma.j.cn115989-20240304-00063.

ABSTRACT                                                                  [Download PDF] [Read Full Text

Objective  To investigate the effect and mechanism of the V-set and immunoglobulin domain-containing 4 ( VSIG4 ) gene mutation on the function of microglia in retinitis pigmentosa (RP).

Methods  Localization of VSIG4 in the retina was detected by immunofluorescence.HMC3 cells (human microglial cells) were transfected with wild-type (Len-WT) VSIG4 gene, mutant type (Len-Mut) VSIG4 gene and empty vector virus (Len-Cont) and stimulated by the presence or absence of lipopolysaccharide (LPS), then divided into control group, LPS-Len-Mut group, LPS-Len-WT group, LPS-Len-Cont group, Len-Mut group, Len-WT group and Len-Cont group.The mRNA expression levels of the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected by real-time fluorescence quantitative PCR.Protein expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase 4 (GPX4), nuclear transcription factor-κB (NF-κB) p65 subunit (P65), and phosphorylated P65 (PP65) were detected by Western blot.Cell phagocytic function was detected by phagocytosis assay.Cell migration ability was detected by cell scratch and transwell migration assay.LPS- stimulated HMC3 cells were co-cultured with 661W cells (mouse retinal photoreceptor cells), and the expression levels of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) proteins of the cells were detected by Western blot.The number of apoptotic cells was determined by apoptosis assay.

Results  VSIG4 was localized to microglia in mouse retina.The real-time fluorescence quantitative PCR results showed that compared with LPS-Len-WT group, the relative expression levels of IL-1β and TNF-α mRNA in HMC3 cells were significantly increased in LPS-Len-Mut group (both at P<0.05).The Western blot results showed that compared with LPS- Len-WT group, the protein expression levels of Nrf2, HO-1, and GPX4 in HMC3 cells were significantly reduced in LPS-Len-Mut group, and the PP65/P65 ratio was significantly increased (all at P<0.05).The phagocytic experiment results showed that the phagocytic rates of HMC3 cells in Len-Cont group, LPS-Len-Cont group, LPS-Len-WT group, and LPS-Len-Mut group were (35.67±3.22)%, (63.67±10.07)%, (84.00±3.46)%, and (64.67±2.31)%, respectively, showing a statistically significant difference ( F=59.06, P<0.001).Compared with LPS-Len-WT group, the phagocytic rate of HMC3 cells was significantly reduced in LPS-Len-Mut group ( P<0.05).The results of cell scratch and transwell migration assay showed that compared with LPS-Len-WT group, the migration rate of HMC3 cells at 24 and 48 hours and the number of invading cells per unit area at 24 hours were significantly reduced in LPS-Len-Mut group (all at P<0.05).Compared with LPS-Len-WT group, the expression ratio of Bax/Bcl-2 protein and the number of cell apoptosis were significantly increased in the LPS-Len-Mut group under the co-culture system (both at P<0.05).

Conclusions  VSIG4 is localized to mouse retinal microglia.When the VSIG4 gene in RP mutates, HMC3 cells under LPS stimulation exhibit a series of changes, including activation of the NF-κB signaling pathway, decreased activation of the Nrf2/HO-1 signaling pathway, increased secretion of inflammatory cytokines, reduced phagocytic and migratory abilities, and increased cell apoptosis in co-culture systems.

VSIG4;Genetic mutation;Retinitis pigmentosa;Microglia;Phagocytosis

Authors Info & Affiliations

Xu Chunlong
Department of Ophthalmology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
Zhang Guowei
Department of Ophthalmology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
Du Jun
Department of Ophthalmology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
Jia Zhen
Department of Ophthalmology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
Wang Jingping
Department of Ophthalmology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
Wang Ziwen
Department of Ophthalmology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
Li Yang
Department of Ophthalmology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
Lu Hong
Department of Ophthalmology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
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