Elucidating the mechanism by which Aloe-emodin from Cassiae semen treats diabetic retinopathy via network pharmacology and experimental verification

Figures

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  Fig. 1. Flow chart of the analysis process in the study.

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  Fig. 2. Active pharmaceutical ingredients and targets Cassiae Semen. The blue circle nodes represent the active ingredients of Cassiae Semen. The red circle nodes represent 64 targets related to the active ingredients shown above.

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  Fig. 3. PPI network of common Cassiae Semen-DR targets. (A) Compound-target interaction network. The red nodes represent the 11 active ingredients of Cassiae Semen, and the yellow nodes represent the 21 overlapping targets related to DR. The size of the node indicates its degree value in the network. MOL000471 (aloe-emodin, AE) is highlighted as the most connected compound. (B) PPI network of the 21 common targets constructed using the STRING database. The edges represent functional or physical associations between proteins. (C) Top 10 core genes identified from the PPI network using the CytoHubba plugin in Cytoscape, ranked by the Maximal Clique Centrality algorithm.

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  Fig. 4. GO and KEGG enrichment analyses of the DR-related targets of Cassiae Semen. (A) Significantly enriched biological process terms. (B) Significantly enriched cellular component terms. (C) Significantly enriched molecular function terms. (D) Top 20 significantly enriched KEGG pathways. A-D were sorted according to -log P values (P < 0.05).

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  Fig. 5. Effects of AE on the viability of LPS-stimulated BV2 microglia. BV2 cells were treated with 1 µg/ml LPS in the absence or presence of AE (1, 2, 4, 6, 8 or 10 µM) for 12 h. Cell viability was determined by a CCK-8 assay. All values are presented as the means ± standard deviations (SDs), n = 3, ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. the LPS group.

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  Fig. 6. Effect of AE on the mRNA expression of COX2, iNOS and TNF-α (A) and IL-1β and IL-6 (B) after LPS treatment. The LPS + AE group was compared with the LPS group, and the LPS group was compared with the control group. **P < 0.01. ***P < 0.001, ****P < 0.0001.

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  Fig. 7. Effect of AE on the protein levels of inflammatory cytokines. (A) TNF-α, IL-1β and IL-6 levels were measured via ELISA. (B) COX2 expression was measured via Western blotting. The LPS group was compared with the control group. The LPS + AE group was compared with the LPS group. The data are presented as the means ± SDs (n = 3) and were analyzed by one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.