Hirudo extract ameliorates proliferative vitreoretinopathy by promoting autophagy and attenuating the THBS2/PI3K/Akt pathway

Abstract

Epithelial‒mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells is believed to play a key role in the pathogenesis of proliferative vitreoretinopathy (PVR). The ability of Hirudo to promote blood flow and dispel blood stasis may be related to its anti-EMT effects. Through the use of a network pharmacology method, the mechanism by which Hirudo treats PVR was investigated in this study, and the findings were confirmed through in vitro cellular tests. The targets and pathways of the active compounds of Hirudo against PVR were predicted via a network pharmacology technique. ARPE-19 cells were treated with several doses of Hirudo extract, that did or did not contain TGF-β2 (10 ng/mL). CCK-8, wound healing, and Transwell assays were performed to detect the viability, migration, and invasion of the cells. Immunofluorescence staining was used to detect F-actin expression. Autophagy was observed via transmission electron microscopy. The mRNA expression of MMP9, N-cadherin, vimentin, THBS2, PI3K, and Akt was measured via RT‒qPCR. Western blotting was used to detect the protein expression of MMP9, N-cadherin, vimentin, LC3B, THBS2, PI3K, p-PI3K, Akt, and p-Akt. The prediction yielded a total of 546 potential targets, 875 PVR-associated disease targets, and 22 Hirudo-PVR cross-targets involving VWF, THBS2, TP53, and IGF1R, and it was inferred that the mechanism might be related to the PI3K‒Akt signaling pathway. After APRE-19 cells were treated with TGF-β2, cell migration, invasion, and viability increased. Additionally, the expression of F-actin, MMP9, N-cadherin, vimentin, THBS2, PI3K, p-PI3K, Akt, and p-Akt was upregulated. Hirudo extract counteracted the effects of TGF-β2 among APRE-19 cells. The promotion of autophagy in APRE-19 cells by TGF-β2 is highlighted, as evidenced by an increase in the LC3II/LC3I ratio. The autophagy-promoting effect of TGF-β2 on APRE-19 cells was further enhanced by Hirudo extract. Hirudo extract improved PVR by promoting autophagy and inhibiting the EMT process, and the mechanism may be related to the regulation of the THBS2/PI3K/Akt pathway.

Keywords: Epithelial–mesenchymal transition; Hirudo extract; Proliferative vitreoretinopathy; Retinal pigment epithelial; THBS2/PI3K/Akt abstract.

Fig. 1

Network pharmacology analysis of the active components of Hirudo. (A) Venn diagram of disease-active component intersection targets. (B) PPI network. (C) GO enrichment analysis. (D) KEGG enrichment analysis. BP = biological process, CC = cell composition, MF = molecular function, GO = gene ontology, KEGG = Kyoto Encyclopedia of Genes and Genomes.(For previous uses, the Kanehisa laboratory have happily provided permission.)

Fig. 2

Determination of the Hirudo extract concentration. (A) CCK-8 assay was used to detect the effects of different concentrations of Hirudo extract (0, 50, 100, 200, 400, 800, and 1600 µg/mL) on the viability of ARPE-19 cells. (B) Wound healing assays were used to determine how various Hirudo extract concentrations (0, 50, 100, 200, and 400 µg/mL) affect ARPE-19 cell migration. (C) CCK-8 assay was used to measure the impact of various Hirudo extracts (0, 50, 100, 200, 400, 800, and 1600 µg/mL) on the viability of TGF-β2-induced ARPE-19 cells. (D) Wound healing assay to determine how different Hirudo extract concentrations (0, 50, 100, 200, and 400 µg/mL) affect TGF-β2-induced ARPE-19 cell migration. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the TGF-β2 group.

Fig. 3

Effects of Hirudo extract on the invasion of TGF-β2-induced ARPE-19 cells. (A) ARPE-19 cells were tested for proliferative viability via the CCK-8 assay. (B) ARPE-19 cell invasion capacity was investigated via a Transwell assay. ***P < 0.001 vs. the control group; #P < 0.05, ###P < 0.001 vs. the TGF-β2 group; @@P < 0.01, @@@P < 0.001, vs. the TGF-β2 + 400 µg/mL Hirudo extract group; $P < 0.05 vs. the TGF-β2 + 800 µg/mL Hirudo extract group.

Fig. 4

Effects of Hirudo extract on cytoskeletal F-actin polymerization in TGF-β2-induced ARPE-19 cells. F-actin expression (red immunofluorescence) was detected via immunofluorescence staining (original magnification: ×400). DAPI was used to stain the cell nucleus (blue). ***P < 0.001 vs. the control group; ###P < 0.001 vs. the TGF-β2 group; @@P < 0.01 vs. the TGF-β2 + 400 µg/mL Hirudo extract group; $P < 0.05 vs. the TGF-β2 + 800 µg/mL Hirudo extract group.

Fig. 5

Effect of Hirudo extract on EMT in TGF-β2-induced ARPE-19 cells. (A) Western blotting was used to detect the protein expression of MMP9, N-cadherin, and vimentin. (B) The mRNA expression of MMP9, N-cadherin, and vimentin was determined via RT‒qPCR. ***P < 0.001 vs. the control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the TGF-β2 group; @P < 0.05, @@P < 0.01 vs. the TGF-β2 + 400 µg/mL Hirudo extract group; $P < 0.05 vs. the TGF-β2 + 800 µg/mL Hirudo extract group.

Fig. 6

Effect of Hirudo extract on autophagy in TGF-β2-induced ARPE-19 cells. (A) Autophagy was observed via transmission electron microscopy (original magnification: ×20000). Mitochondria (Mi), nucleus (N), mild mitochondrial swelling (red arrow), and autophagy (green arrow) are shown. (B) Western blot analysis of LC3B protein expression. The LC3II/LC3I ratio was calculated. **P < 0.01 vs. the control group; ##P < 0.01 vs. the TGF-β2 group; @P < 0.05, vs. the TGF-β2 + 400 µg/mL Hirudo extract group.

Fig. 7

Effects of Hirudo extract on the THBS 2/PI3K/Akt pathway in TGF-β2-induced ARPE-19 cells. (A) Western blotting was used to detect the protein expression of THBS2, PI3K, p-PI3K, Akt, and p-Akt. (B) The mRNA expression of THBS2, PI3K, and Akt was determined through RT-qPCR. ***P < 0.001 vs. the control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the TGF-β2 group; @P < 0.05, @@P < 0.01 vs. the TGF-β2 + 400 µg/mL Hirudo extract group; $P < 0.05, $$P < 0.01 vs. the TGF-β2 + 800 µg/mL Hirudo extract group.