GSDMD mediated pyroptosis induced inflammation of Graves' orbitopathy via the NF-κB/ AIM2/ Caspase-1 pathway.

Gasdermin D (GSDMD) is a key executor which triggers pyroptosis as well as an attractive checkpoint in various inflammatory and autoimmune diseases but it has yet to prove its function in Graves'orbitopathy (GO). Our aim was to investigate GSDMD levels in orbital connective tissue and serum of GO patients and then assess the association between serum levels and patients' clinical activity score (CAS). Further, GSDMD-mediated pyroptosis and the underlying mechanism in inflammatory pathogenesis in the cultured orbital fibroblasts (OFs) of GO patients were examined. OFs were collected after tumor necrosis factor (TNF)-α or interferon (IFN)-γ treatment or combination treatment at different times, and the expression of GSDMD and related molecular mechanisms were analyzed. Then, we constructed the GSDMD knockout system with siRNA and the system was further exposed to the medium with or without IFN-γ and TNF-α for a specified time. Finally, we evaluated the production of interleukin (IL)-1ß and IL-18. We found that serum GSDMD levels were elevated and positively correlated with the CAS in GO patients. Meanwhile, the expression of GSDMD and N-terminal domain (NT-GSDMD) in orbital connective tissue of GO patients was augmented. Also, increased expression of GSDMD and related pyroptosis factors was observed in vitro model of GO. We further demonstrated that GSDMD-mediated pyroptosis induced inflammation via the nuclear factor kB (NF-κB)/absent in melanoma-2 (AIM-2)/caspase-1 pathway. In addition, blocking GSDMD suppressed proinflammatory cytokine production in GO. We concluded that GSDMD may be a biomarker as well as a potential target for the evaluation and treatment of inflammation related with GO.

Serelaxin Alleviates Fibrosis in Thyroid-Associated Ophthalmopathy via the Notch Pathway.

Fibrosis is the late stage of thyroid-associated ophthalmopathy (TAO), resulting in serious complications. Effective therapeutic drugs are still lacking. We aimed to explore the mechanism of TAO fibrosis and to find a targeted drug. High-throughput RNA sequencing was performed on orbital connective tissues from twelve patients with TAO and six healthy controls. Protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and we identified the hub gene by Cytoscape software. Additionally, the RNA sequencing results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatic prediction identified the functions of differentially expressed genes (DEGs). Further orbital connective tissue and serum samples of the TAO and control groups were collected for subsequent experiments. Histologic staining, Western blotting (WB), qRT-PCR, enzyme-linked immunosorbent assays (ELISAs), gene overexpression through lentiviral infection or silencing gene by short interfering RNA (siRNA) were performed. We found that the relaxin signaling pathway is an important regulatory pathway in TAO fibrosis pathogenesis. Serelaxin exerts antifibrotic and anti-inflammatory effects in TAO. Furthermore, the downstream Notch pathway was activated by serelaxin and was essential to the antifibrotic effect of serelaxin in TAO. The antifibrotic effect of serelaxin is dependent on RXFP1.

Metformin Attenuates Inflammation and Fibrosis in Thyroid-Associated Ophthalmopathy.

The pathogenesis of thyroid-associated ophthalmopathy (TAO) is still unclear, and therapeutic drugs have great limitations. As metformin has multiple therapeutic effects in many autoimmune diseases, we explored the effects of metformin on TAO in an in vitro fibroblast model. We used orbital connective tissues and fibroblasts that were obtained from TAO patients and normal controls. The activity of adenosine monophosphate-activated protein kinase (AMPK) and the levels of inflammatory or fibrotic factors were examined by immunofluorescence (IF) and immunohistochemistry (IHC). Quantitative real-time polymerase chain reaction (qPCR), cytokine quantification by enzyme-linked immunosorbent sssay (ELISA), IF, and western blotting (WB) were used to measure the expression of factors related to inflammation, fibrosis, and autophagy. To determine the anti-inflammatory and antifibrotic mechanisms of metformin, we pretreated cells with metformin, 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR, an AMPK activator) or compound C (CC, an AMPK inhibitor) for 24 h and used WB to verify the changes in protein levels in the AMPK/mammalian target of rapamycin (mTOR) pathway. We determined that the low activity of AMPK in the periorbital tissue of TAO patients may be closely related to the occurrence and development of inflammation and fibrosis, and metformin exerts multiple effects by activating AMPK in TAO. Furthermore, we suggest that AMPK may be a potential target of TAO therapy.

Differential Circular RNA Expression Profiling of Orbital Connective Tissue From Patients With Type I and Type II Thyroid-Associated Ophthalmopathy.

Purpose: The purpose of this study was to investigate the molecular mechanism underlying thyroid-associated ophthalmopathy (TAO) clinical subtypes, to do so, we performed transcriptomic analysis to reveal the expression profile of circular RNAs (circRNAs) in TAO subtypes. Methods: High-throughput RNA-sequencing was performed in six pairs of type I and type II orbital connective tissue samples from patients with TAO. The expression levels of circRNAs and mRNAs in type I and type II samples were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in another three pairs of type I and type II TAO connective tissue samples. We used bioinformatics predictions to construct a circRNA-microRNA (miRNA)-mRNA network. A protein-protein interaction (PPI) network was constructed based on differential mRNA expression, and the hub genes were determined by the Cytoscape software plugin. Functional and pathway enrichment analyses were performed to elucidate circRNA function. Lentiviral-mediated overexpression of hsa_circ_0007006 and the relationship between hsa_circ_0007006 with COL1A1 and MMP2 were evaluated by Western blotting (WB). Moreover, the differential pathways were assessed by WB. Results: RNA sequencing results predicted a total of 7489 circRNAs and 15,803 mRNAs, with 94 upregulated and 76 downregulated circRNAs and 488 upregulated and 138 downregulated mRNAs. The qRT-PCR analysis of seven dysregulated circRNAs and two major mRNAs validated the RNA-sequencing data. The competing endogenous RNA (ceRNA) network included 7 circRNAs, 23 miRNAs, and 262 mRNAs. Functional analysis revealed several important pathways. Overexpression of hsa_circ_0007006 led to decreased expression levels of COL1A1 and MMP2. Activation of the relaxin signaling pathway differed between the two subtypes. Conclusion: We showed that circRNAs are differentially expressed between type I and type II TAO. We speculate that the hsa_circ_0007006-COL1A1 and MMP2-relaxin signaling pathways are important regulatory axes in the pathogenesis of this disease type and could be considered promising diagnostic and therapeutic targets in the future.

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

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-ß1-induced expression of fibrosis markers, including alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF). Furthermore, DHA inhibited TGF-ß1 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-1ß 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.

Disulfiram Exerts Antifibrotic and Anti-Inflammatory Therapeutic Effects on Perimysial Orbital Fibroblasts in Graves' Orbitopathy.

Fibrosis of extraocular muscles (EOMs) is a marker of end-stage in Graves' orbitopathy (GO). To determine the antifibrotic and anti-inflammatory therapeutic effects and the underlying molecular mechanisms of disulfiram (DSF) on perimysial orbital fibroblasts (pOFs) in a GO model in vitro, primary cultures of pOFs from eight patients with GO and six subjects without GO (NG) were established. CCK-8 and EdU assays, IF, qPCR, WB, three-dimensional collagen gel contraction assays, cell scratch experiments, and ELISAs were performed. After TGF-ß1 stimulation of pOFs, the proliferation rate of the GO group but not the NG group increased significantly. DSF dose-dependently inhibited the proliferation, contraction, and migration of pOFs in the GO group. Additionally, DSF dose-dependently inhibited fibrosis and extracellular matrix production markers (FN1, COL1A1, α-SMA, CTGF) at the mRNA and protein levels. Furthermore, DSF mediates antifibrotic effects on GO pOFs partially through the ERK-Snail signaling pathway. In addition, DSF attenuated HA production and suppressed inflammatory chemokine molecule expression induced by TGF-ß1 in GO pOFs. In this in vitro study, we demonstrate the inhibitory effect of DSF on pOFs fibrosis in GO, HA production, and inflammation. DSF may be a potential drug candidate for preventing and treating tissue fibrosis in GO.

Characterisation of macular superficial vessel density alteration in preclinical ethambutol-induced optic neuropathy using optical coherence tomography angiography.

AIM: To investigate the changes in macular vessel density (mVD) and its relationship to macular ganglion cell-inner plexiform layer (mGCIPL) thickness in patients receiving ethambutol (EMB) therapy for tuberculosis without recognisable clinical symptoms or signs of EMB-induced optic neuropathy (EON). METHODS: A total of 23 eyes of 13 patients using EMB therapy for 6 months without EON (preclinical EON) as the EMB group, 40 eyes of 23 healthy individuals as the normal control group and 18 eyes of 10 patients with tuberculosis before receiving EMB therapy as the blank control group were retrospectively analysed. The mean peripapillary retinal nerve fibre layer (pRNFL) and mGCIPL thicknesses and mVD were measured using optical coherence tomography angiography. Patients in the EMB group were compared with individuals in the normal and blank control groups, and changes in macular parameters were evaluated. RESULTS: Central circle mVD (cCVD) was significantly lower in the EMB group than in both control groups (generalised estimating equation (GEE), p=0.003 and 0.029, respectively). The mGCIPL thickness in all regions and the mean pRNFL thickness were not significantly different between the EMB group and both control groups (GEE, p=1.000 for all). There were no significant differences in mVD, mGCIPL thickness and mean pRNFL thickness between the normal control and blank control groups (p>0.05). In the generalised linear model analyses, the minimum and inferonasal mGCIPL thicknesses were positively correlated with cCVD in the EMB group (ß=1.285, p=0.003 and ß=0.770, p=0.024, respectively). CONCLUSIONS: cCVD decreased with no changes in mGCIPL and mean pRNFL thicknesses in patients with preclinical EON. The minimum and inferonasal mGCIPL thicknesses were positively correlated with cCVD. cCVD might be an early indicator for monitoring early-stage EMB toxicity.

Relationship between axial length and spherical equivalent refraction in Chinese children.

Purpose: To evaluate the relationship between axial length (AL) and spherical equivalent refraction (SER) in Chinese children. Methods: This hospital-based cross-sectional study included 1208 eyes (from 617 Chinese boys and 591 Chinese girls), ranging between 2 and 12 years. All subjects were divided into subgroups according to the spherical equivalent refraction (SER) (hyperopia, emmetropia, and myopia) and age (2-6 years, 7-9 years, and 10-12 years). Comparisons were made between age, sex, and SER groups. Multiple linear regression analysis was used to evaluate the correlation of AL and SER for all groups. Results: The mean AL and SER were significantly different among the three age groups: 2-6 years group (AL: 22.24 â€‹± â€‹1.59 â€‹mm; SER: 0.73 â€‹± â€‹2.67 D); 7-9 years group (AL: 23.49 â€‹± â€‹1.10 â€‹mm; SE: -0.68 â€‹± â€‹1.97 D) and 10-12 years group (AL: 24.33 â€‹± â€‹1.02 â€‹mm; SER: -1.72 â€‹± â€‹1.86 D). Boys showed longer AL compared with the girls (23.66 â€‹± â€‹1.51 â€‹mm vs 23.05 â€‹± â€‹1.32 â€‹mm). However, as for SER, the girls (-0.70 â€‹± â€‹2.17 D) showed smaller SER (more myopia) compared with the boys (-0.4 â€‹± â€‹2.48 D). After adjusting for age and sex, the SER tended to decrease (became more myopic) 1.23 D (95% CI: 1.15-1.30D) with a 1 â€‹mm increase of AL. Among the different SER groups, the SER tended to become more myopic per mm of AL by 1.09 D (95% CI: 0.97-1.21D) for the myopia group, 1.38 D (95% CI: 1.23-1.54 D) for the hyperopia group, and 0.05 D (95% CI: 0.02-0.08 D) for the emmetropic group. In addition, an increase of 1 â€‹mm elongation of AL showed a decrease of SER by 1.05 D (95% CI: 0.90-1.20 D) among 2 - 6-year-olds, by 1.40 D (95% CI: 1.30-1.51 D) among 7 - 9-year-olds, and by 1.37 D (95% CI: 1.21-1.52 D) among 10-12-year-olds. As for sexual differences, the girls 1.68 D, (95% CI: 1.57-1.79 D) showed a more significant myopic shift of SER with a 1 â€‹mm increase of AL compared with the boys (0.94 D, 95% CI: 0.84-1.04 D). Conclusions: Our results indicated a strong linear relationship between SER and AL and an early-rising trend of myopia in Chinese children.