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. 2024 Apr 18;17(4):625-637.
doi: 10.18240/ijo.2024.04.04. eCollection 2024.

Neuroprotective effects of acteoside in a glaucoma mouse model by targeting Serta domain-containing protein 4

Hui-Jie Hao  1 , Ya-Hong Li  1 , Bo Yu  1 , Xun Liu  1 , Yan Zhang  1 , Xiao-Li Xing  1
Affiliations

Neuroprotective effects of acteoside in a glaucoma mouse model by targeting Serta domain-containing protein 4

Hui-Jie Hao et al. Int J Ophthalmol. .

Abstract

Aim: To explore the therapeutic effect and main molecular mechanisms of acteoside in a glaucoma model in DBA/2J mice.

Methods: Proteomics was used to compare the differentially expressed proteins of C57 and DBA/2J mice. After acteoside administration in DBA/2J mice, anterior segment observation, intraocular pressure (IOP) monitoring, electrophysiology examination, and hematoxylin and eosin staining were used to analyze any potential effects. Immunohistochemistry (IHC) assays were used to verify the proteomics results. Furthermore, retinal ganglion cell 5 (RGC5) cell proliferation was assessed with cell counting kit-8 (CCK-8) assays. Serta domain-containing protein 4 (Sertad4) mRNA and protein expression levels were measured by qRT-PCR and Western blot analysis, respectively.

Results: Proteomics analysis suggested that Sertad4 was the most significantly differentially expressed protein. Compared with the saline group, the acteoside treatment group showed decreased IOP, improved N1-P1 wave amplitudes, thicker retina, and larger numbers of cells in the ganglion cell layer (GCL). The IHC results showed that Sertad4 expression levels in DBA/2J mice treated with acteoside were significantly lower than in the saline group. Acteoside treatment could improve RGC5 cell survival and reduce the Sertad4 mRNA and protein expression levels after glutamate injury.

Conclusion: Sertad4 is differentially expressed in DBA/2J mice. Acteoside can protect RGCs from damage, possibly through the downregulation of Sertad4, and has a potential use in glaucoma treatment.

Keywords: Serta domain-containing protein 4; acteoside; glaucoma; mice; proteomics.

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Conflict of interest statement

Conflicts of Interest: Hao HJ, None; Li YH, None; Yu B, None; Liu X, None; Zhang Y, None; Xing XL, None.

Figures

Figure 1
Figure 1. The three groups were the C57 mice group, the DBA mice with low IOP (DBA-L) group and the DBA mice with high IOP (DBA-H) group respectively
A: The IOP of three groups of mice from 20 to 26wk; B: The IOP of three groups of mice from 27 to 36wk; C–K: Anterior segment status of three groups of mice at 20, 28, and 36wk. The three groups were C57 mice group (C–E), DBA-L group (F–H), and DBA-H group (I–K) respectively. n=10. The mean IOP of the DBA-L group eyes and the DBA-H eyes was 12.3±0.8 and 16.5±0.5 mm Hg, respectively. IOP: Intraocular pressure; SEM: Standard error of the mean.
Figure 2
Figure 2. Proteomics analysis between DBA-H and normal control groups
A–C: Gene ontology analysis of significantly expressed proteins in DBA-H group compared with normal control group. GO analysis mainly includes three aspects: biological process, cellular component, and molecular function. D: Analysis of the KEGG pathway. E: Top 20 up-regulated expression proteins. F: Top 20 down-regulated expression proteins. G: The number of significantly regulated protein. DBA-H: DBA mice with high intraocular pressure; KEGG: Kyoto Encyclopedia of Genes and Genomes.
Figure 3
Figure 3. Gene ontology analysis of significantly differentially expressed proteins between DBA-H group and DBA-L group, including biological process (A), cellular component, cellular component (B), and molecular function (C).
DBA-H: DBA mice with high IOP; IOP: Intraocular pressure; DBA-L: DBA mice with low IOP.
Figure 4
Figure 4. IOP and anterior segment of mice over 4wk observation time
A: Molecular structures of acteoside; B: Weight of mice at different times; C: The amount of gavage at different times; D: The IOP in three groups of mice over 4wk observation time. The mean IOP of the DBA-L group eyes and the DBA-H eyes was 17.0±0.4 and 14.9±0.5 mm Hg, respectively (P<0.001). E: Effects of gavage on the anterior segment of mice over 4wk observation time. Error bars indicate SEM. IOP: Intraocular pressure; SEM: Standard error of the mean; DBA-H: DBA mice with high IOP; DBA-L: DBA mice with low IOP.
Figure 5
Figure 5. The histological analysis of mice eyes at 40wk
The three groups were respectively normal control group (A), saline group (B) and acteoside group (C). D: Histological analysis GCL counts in retinas. E: The thickness of each layer of the retina in the three groups of mice. aP<0.05; bP<0.01, cP<0.001, dP<0.0001. Error bars indicate SEM. GCL: Ganglion cell layer; IPL: Inner plexiform layer; INL: Inner nuclear layer; OPL: Outer plexiform layer; ONL: Outer nuclear layer; OS: Outer segment; SEM: Standard error of the mean.
Figure 6
Figure 6. The retinal function examinations of mice eyes at 40wk
A: VEP tests waves of normal control group, saline group and acteoside group. B-D: The amplitude of N1 (B), P1 (C), N1-P1 (D) in three groups. E: Waveforms of dark-adapted 0.01 ERG (DA 0.01 ERG) tests, dark-adapted 3.0 ERG (DA 3.0 ERG) tests and dark-adapted 30.0 ERG (DA 30.0 ERG) tests for normal control, saline and acteoside group. F: A-wave amplitudes recorded in DA 0.01, DA 3, DA 30.0 ERG tests. G: B-wave amplitudes recorded in DA 0.01, DA 3, DA 30.0 ERG tests. The aforementioned 0.01, 3.0 and 30.0 respectively represented 0.01, 2.562, and 25.62 cd·s/m2 stimulus in ERG tests. bP<0.01, cP<0.001, dP<0.0001. Error bars indicate SEM. VEP: Visual evoked potential; DA: Dark adaptation; ERG: Electroretinogram; LA: Light adaptation; SEM: Standard error of the mean.
Figure 7
Figure 7. The immunohistochemistry of mice eyes at 40wk
Expression of Sertad4 in normal control group (A), saline group (B) and acteoside group (C). D: Statistics of the average optical density values of the three groups. E: Statistics of the average optical density values of the three groups of GCL, INL, and ONL. aP<0.05; bP<0.01, cP<0.001, dP<0.0001. Error bars indicate SEM. GCL: Ganglion cell layer; INL: Inner nuclear layer; ONL: Outer nuclear layer; SEM: Standard error of the mean.
Figure 8
Figure 8. Acteoside relieves cell injury in RGC5 via Sertad4
A: Three groups of cells were observed under a microscope to assess cell number and growth status; B: Cell viability of the three groups. C: The relative expression levels of Sertad4 genes in three group cells. D: Western blot representatives of Sertad4 and GAPDH in three groups. Two replicate wells were set for each group. E: Ratio of Sertad4 over GAPDH in three groups. aP<0.05; bP<0.01, cP<0.001, dP<0.0001. Error bars indicate SEM. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; SEM: Standard error of the mean; RGC5: Retinal ganglion cell 5.
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