Eye Drops of Metformin Prevents Fibrosis After Glaucoma Filtration Surgery in Rats via Activating AMPK/Nrf2 Signaling Pathway

Abstract

Metformin has effective therapeutic effects in anti-tumor and anti-fibrotic diseases. However, how the antifibrotic effect of metformin in the eye and how it is transferred are still unclear. Here, the eye drop of metformin treatment was studied in Sprague-Dawley (SD) rats of glaucoma filtrating surgery (GFS). Rats were administered randomly bilateral drops: control group (without surgery), GFS group, metformin group or mitomycin C (MMC) group (sponge application intraoperatively, 0.02%). Bleb features and intraocular pressure (IOP) were assessed for postoperative week 4. Metformin effectively inhibited fibrosis and improved the surgical outcomes of GFS. In vitro, we found that the degree of oxidative stress and fibrosis in metformin pretreated-Human Conjunctival Fibroblasts (HConFs) were reduced; the pro-fibrotic response of HConFs were decreased by inducing macrophagic polarity changes. Besides, the inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2)/AMP-activated protein kinase (AMPK) and the competition of organic cation transporters (OCTs) effectively reduced the anti-fibrotic capability of metformin. Together, this experiment indicates that metformin enters into HConFs cell with OCTs, which can protect against filtrating blebs scar formation in SD rats of GFS via activating AMPK/Nrf2 axis and the downregulation of profibrogenic and inflammatory biomarkers.

Keywords: AMPK/Nrf2; fibrosis; glaucoma filtration surgery (GFS); macrophages; metformin; organic cation transporters (OCTs); oxidative stress.

Figure 1

Metformin prolongs filtering blebs survival in SD rats of GFS. (A) Schematic illustration of the experimental animal timeline. (B) Quantization of bleb area was analyzed. (C) H&E and (D) Masson’s trichrome were assessed for the fibrotic levels of filtrating blebs. (E–G) Immunohistochemical staining images showing the expression of collagen-1, vimentin, α-SMA and OPN in filtrating bleb tissues at postoperative day 14. n = 10. Data shown as mean values ±  SD. *** denotes p < 0.001.

Figure 2

AICAR inhibits TGF-β2-induced HConFs fibrosis. (A) The expression of Thr172 AMPK phosphorylation in filtrating blebs tissues in rats were analyzed by historical images. (B, C) The fibrotic levels in TGF-β2 treated HConFs followed by AICAR treatment were determined by western blotting/qRT-PCR. (D) The ROS levels were analyzed by flow cytometry assay. (E) Relative antioxidants proteins and (F) genes were determined. (G) The migration capabilities of HConFs were assessed by transwells. n = 3. All data shown as mean values ± SD. ** and *** denote p < 0.01 and p < 0.001, respectively.

Figure 3

Metformin reduces TGF-β2-induced HConFs fibrosis. (A, B) The anti-fibrotic effects of metformin on TGF-β2 treated HConFs were analyzed by western blot/qRT-PCR. (C, D) The antioxidative effect of metformin on TGF-β2-induced HConFs was examined. (E) The ROS levels of HConFs were assessed by flow cytometry. (F) The migration capability of TGF-β2-induced HConFs followed by metformin treatment were analyzed by transwells and statistical analysis. n = 3. All data shown as mean values ± SD. ** and *** denote P < 0.01 and P < 0.001, respectively.

Figure 4

Metformin induces cell cycle arrest at G0/G1 phase in HConFs. (A) Cell cycle was analyzed by flow cytometry assay and (B) quantization of the rates in HConFs cells. (C) CCK8 was used to analyze the proliferation of HConFs. (D) The genes levels of CDK4, P21, CyclinD1 and P27 were assessed by qRT-PCR. (E) The relative cell cycle proteins level of CDK4, CyclinD1, P21 and P27 were tested by Western blotting. **p < 0.01, ^###/** p < 0.001. MET = Metformin.

Figure 5

Metformin reduces fibrosis in HConFs pretreated by TGF-β2 via activating AMPK/Nrf2 axis. (A–B) AICAR or metformin could increase the levels of p-Nrf2(S40), Nrf2 and p-AMPK in HConFs after TGF-β2 precondition for 24 h. (C) Cell cycle was tested and quantization of the rates in HConFs cells. Compound C or ML385 could reverse the inhibition of metformin on HConFs’ proliferation. (D–F) Cell cycle relative proteins/genes (CDK4, CyclinD1, P21 and P27) were detected by qRT-PCR/western blotting. (G–I) Antioxidant defense relative protein/genes γ-GCS, SOD1/2 and (J–L) fibrotic proteins/genes collagen-1, fibronectin, α-SMA level in HConFs treated by metformin with/without Compound C (10 μmol/L) or ML385 (10 mM) treatment for 24 h. MET = metformin, n = 3. All data shown as mean values ± SD. **^/## and ***^/### denote P < 0.01 and P < 0.001, respectively.

Figure 6

The inhibitor of OCTs could reduce the capability of anti-proliferation of metformin. (A) Cell cycle was assessed by flow cytometry assay and (B) quantization of the rates in HConFs. (C–E) Cell cycle relative proteins/genes (CyclinD1, CDK4, P21 and P27) level were assessed in HConFs after Atropine/Quinidine treatment by qRT-PCR and western blot. (F–H) Relative fibrotic proteins/genes were assessed. (I–K) Antioxidants related proteins and genes (α-SMA, collagen-1, fibronectin, N-cadherin and vimentin) were assessed in HConFs after Atropine/Quinidine treatment by qRT-PCR or western blotting. MET = metformin, n = 3. Data shown as mean values ± SD. *^/#p < 0.05, **^/##p < 0.01, ***^/###p < 0.001.

Figure 7

Metformin reduces the fibrotic levels of HConFs via regulating macrophage polarization. (A, B) Immunofluorescence co-staining of α-SMA and CD 206/iNOS in filtrating blebs tissues. (C–E) The fibrosis or proliferation relative genes and proteins level of HConFs co-cultured with macrophage. (F) Quantification of iNOS, IL-6, TNF-α and IL-1β in M1-polarized macrophages (or M2-polarized macrophages) with/without metformin precondition by qRT-PCR. (G) The migration capability of HConFs co-cultured with M0, M1- and M2-polarized macrophages followed by metformin treatment were analyzed by transwells. MET = metformin. Data shown as mean values ± SD. n = 3, ***p < 0.001.

Figure 8

Schematic illustration of the mechanism of metformin reduces oxidative stress and decreases proliferation of HConFs against fibrosis. Briefly, metformin obviously promotes antioxidant defense, inhibits proliferation via activating AMPK/Nrf2 axis and the downregulation of inflammatory biomarkers to improves issues repair potential.