Dihydroartemisinin Exerts Antifibrotic and Anti-Inflammatory Effects in Graves' Ophthalmopathy by Targeting Orbital Fibroblasts

Abstract

Graves' ophthalmopathy (GO) is a common orbital disease that threatens visual function and appearance. Orbital fibroblasts (OFs) are considered key target and effector cells in GO. In addition, hyaluronan (HA) production, inflammation, and orbital fibrosis are intimately linked to the pathogenesis of GO. In this study, we explored the therapeutic effects of dihydroartemisinin (DHA), an antimalarial drug, on GO-derived, primary OFs. CCK8 and EdU assays were applied to evaluate the antiproliferative effect of DHA on OFs. Wound healing assays were conducted to assess OF migration capacity, while qRT-PCR, western blotting, ELISA, and immunofluorescence were used to determine the expression of fibrosis-related and pro-inflammatory markers in these cells. Moreover, RNA sequencing was conducted to identify differentially expressed genes (DEGs) in DHA-treated OFs, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs was performed to explore potential mechanisms mediating the antifibrotic effect of DHA on GO-derived OFs. Results showed that DHA dose-dependently inhibited OF proliferation and downregulated, at the mRNA and protein levels, TGF-β1-induced expression of fibrosis markers, including alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF). Furthermore, DHA inhibited TGF-β1 induced phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3), which suggested that DHA exerted antifibrotic effects via suppression of the ERK and STAT3 signaling pathways. In addition, DHA suppressed the expression of pro-inflammatory cytokines and chemokines, including IL-6, IL-8, CXCL-1, MCP-1, and ICAM-1, and attenuated HA production induced by IL-1β in GO-derived OFs. In conclusion, our study provides first-time evidence that DHA may significantly alleviate pathogenic manifestations of GO by inhibiting proliferation, fibrosis- and inflammation-related gene expression, and HA production in OFs. These data suggest that DHA may be a promising candidate drug for treatment of GO.

Keywords: ERK; Graves’ ophthalmopathy; STAT3; dihydroartemisinin; fibrosis; inflammation; orbital fibroblast.

Figure 1

Effect of DHA on the cellular viability of OFs from GO and non-GO patients. (A) OFs obtained from 3 GO patients and 3 non-GO patients were treated with increasing concentrations of DHA (0, 2.5, 5, 10, 20, 40, and 80 μM) for 24, 48, and 72 h, respectively. Cell viability is presented as the percentage relative to the viability of the untreated cells. (B, D) Representative images of the EdU assay results in GO OFs and non-GO OFs treated with different concentrations of DHA (10, 20 μM). The cells were observed using a fluorescence microscope, scale bars = 50 μm. Green: EdU, Blue: 4′,6-diamidino-2-phenylindole (DAPI). (C, E) Quantification of the EdU assay results of GO OFs and non-GO OFs(Ctrl, DHA [10, 20 μM], n = 5). For (A, C), and (E), the summarized data are reported as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001, versus the Ctrl group.

Figure 2

DHA exerts antifibrotic effects on OFs. (A, B) The mRNA levels of α-SMA, CTGF, COL1A1, and FN1 in GO and non-GO OFs (n = 3). (C) The protein expression levels of α-SMA and CTGF were determined using western blot analysis (n = 3). (D) The densities of α-SMA and CTGF protein bands were quantified and normalized to β-Tubulin. (E) Representative images of α-SMA and COL1A1 immunostaining in GO OFs treated with TGF-β1 (10 ng/mL, 48 h) with or without pretreatment of DHA (20 μM, 3 h). The cells were observed using a confocal microscope. Scale bars = 25 μm. Red: α-SMA, Green: COL1A1, Blue: DAPI. (F) Quantification of mean idensity of α-SMA and COL1A1. For (A, B, D, F), the summarized data are reported as the mean ± standard error of the mean. ^#P < 0.05, ^{# #}P < 0.01, ^{# # #}P < 0.001, ^{# # # #}P <0.0001 compared with the control; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with TGF-β1 alone. ns denotes no statistical significance versus the control/TGF-β1.

Figure 3

DHA suppresses the migration capacity of GO-derived OFs. (A) Representative images of wound repair ability of GO OFs (n = 3). Scale bar = 50 μm. (B) Statistical analysis of the rate of wound closure. ^{# # #}P < 0.001 compared with the control; *P < 0.05, ****P < 0.0001 compared with TGF-β1 alone. ns denotes no statistical significance versus the TGF-β1.

Figure 4

DHA suppresses TGF-β1-induced phosphorylation levels of ERK1/2 and STAT3. (A) Differentially expressed genes (DEGs) were explored and presented in heatmap of mRNA abundance. Columns denoting GO OFs grouped by TGF-β1(10 ng/mL, 48 h) stimulation with or without a 3-h pretreatment of DHA (20 μM) were sorted by diversity within the TGF-β1 group (n=3) and the TGF-β1+DHA group (n=3); Rows denoting RNA expression according to their enrichment in TGF-β1 versus TGF-β1+DHA. (B, C) Bubble chart from GO analysis or KEGG analysis showed TGF-β1-stimulated genes were mainly associated with cell differentiation, migration, and extracellular matrix organization. (D) Bubble chart from GO-KEGG analysis showed DHA significantly downregulates fibrosis-associated processes. The MAPK signaling and ERK1/2 signaling were decreased in TGF-β1+DHA group. (E) The top 20 transcription factors differentially regulated by DHA treatment were analyzed by TRRUST. (F) The protein levels of p-ERK, ERK, p-STAT3, STAT3, GAPDH in GO OFs stimulated with TGF-β1 (10 ng/mL, 3 h) with or without a 3-h pretreatment with DHA (10, 20 μM) were determined using western blot analysis (n = 3). (G) The protein levels of p-ERK and p-STAT3 were quantified and analyzed. ^#P < 0.05 compared with the control; *P < 0.05, **P < 0.01 compared with TGF-β1 alone.

Figure 5

DHA exerts an antifibrotic effect on GO-derived OFs by repressing activation of the MAPK/ERK and STAT3 signaling pathways. (A, B) GO-derived OFs were stimulated with TGF-β1 (10 ng/mL, 3 h) with or without a 3-h pretreatment with DHA (20 μM) or ERK inhibitor U0126 (10 μM) or STAT3 inhibitor Stattic (10 μM). The expression and phosphorylation levels of ERK1/2 and STAT3 were determined using western blot analysis (n = 3). (C) The protein levels of pERK1/2 and pSTAT3 were quantified and analyzed. (D) GO-derived OFs were stimulated with TGF-β1 (10 ng/mL, 48 h) with or without a 3-h pretreatment with DHA (20 μM) or ERK inhibitor U0126 (10 μM). The expression levels of α-SMA and CTGF were determined using western blot analysis (n = 3). (E) The protein levels of α-SMA and CTGF were quantified and analyzed. (F) GO-derived OFs were stimulated with TGF-β1 (10 ng/mL,48 h) with or without a 3-h pretreatment with DHA (20 μM) or STAT3 inhibitor Stattic (10 μM). The expression levels of α-SMA and CTGF were determined using western blot analysis (n = 3). (G) The protein levels of α-SMA and CTGF were quantified and analyzed. For (C, E, G), the summarized data are reported as the mean ± standard error of the mean. ^{# #}P < 0.01, ^{# # #}P < 0.001, ^{# # # #}P < 0.0001 compared with the control; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with TGF-β1 alone.

Figure 6

DHA inhibits IL-1β-Induced inflammation and attenuates HA production by OFs. GO OFs and non-GO OFs were stimulated with IL-1β (1 ng/mL,24 h) with or without a 3-h pretreatment with DHA (10, 20 μM). (A, B) The mRNA levels of IL-6, IL-8, CXCL1, MCP-1, and ICAM-1 were detected by qRT-PCR (n = 3). (C, D) The mRNA expression levels of HAS1 and HAS3 were detected by qRT-PCR (n = 3). (E) The concentrations of HA in the GO-derived cell culture supernatants were quantified by ELISA (n = 4). For (A–E), the summarized data are reported as the mean ± standard error of the mean. ^{# # # #}P < 0.0001 compared with the control; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with IL-1β alone. ns denotes no statistical significance versus the control/TGF-β1.