Department of Ophthalmology, Third Xiangya Hospital, Central South University, Changsha, 410013, Hunan, China.
Department of Ophthalmology, Second Xiangya Hospital, Central South University, Changsha, China.
J Endocrinol Invest. 2023 Oct;46(10):2005-2016. doi: 10.1007/s40618-023-02033-3. Epub 2023 Feb 27.
In diagnosing the pathogenesis of Graves' orbitopathy (GO), there is a growing interest in fibrosis generated by orbital fibroblasts (OFs); nevertheless, the involvement of ceruloplasmin (CP) in OFs remains unknown.
Differentially expressed genes (DEGs) were identified through bioinformatic analysis. OFs were isolated from orbital tissue and identified with immunofluorescent staining. The levels of DEGs were validated in GO tissue samples and TGF-β-challenged OFs, and CP was selected for the following laboratory investigations. CP overexpression or knockdown was achieved, and cell viability and fibrosis-associated proteins were investigated to assess the cell phenotype and function. Signaling pathways were subsequently investigated to explore the mechanism of CP function in OFs.
CP and cathepsin C (CTSC) are two overlapped DEGs in GSE58331 and GSE105149. OFs were isolated and identified through fibrotic biomarkers. CP and CTSC were downregulated in GO tissue samples and TGF-β-challenged OFs. CP overexpression or knockdown was achieved in OFs by transducing a CP overexpression vector or small interfering RNA against CP (si1-CP or si2-CP) and verified using a qRT-PCR. CP overexpression inhibited cell viability and reduced the levels of α-SMA, vimentin, fibronectin, and collagen I, whereas CP knockdown exerted opposite effects on OFs. CP overexpression inhibited the phosphorylation of Smad3, Erk1/2, p38, JNK, and AKT; conversely, CP knockdown exerted opposite effects on the phosphorylation of factors mentioned above.
CP was downregulated in GO and suppressed the expression of fibrosis-associated proteins in both GO and normal OFs. CP might serve as a promising therapeutic agent in the treatment regimens for GO.
在诊断格雷夫斯眼病（GO）的发病机制时，人们对眼眶成纤维细胞（OFs）产生的纤维化越来越感兴趣；然而，铜蓝蛋白（CP）在 OFs 中的作用尚不清楚。
通过生物信息学分析鉴定差异表达基因（DEGs）。从眼眶组织中分离 OFs，并通过免疫荧光染色进行鉴定。在 GO 组织样本和 TGF-β 刺激的 OFs 中验证 DEGs 的水平，并选择 CP 进行以下实验室研究。通过过表达或敲低 CP，研究细胞活力和纤维化相关蛋白，以评估细胞表型和功能。随后研究信号通路，以探讨 CP 在 OFs 中的功能机制。
CP 和组织蛋白酶 C（CTSC）是 GSE58331 和 GSE105149 中两个重叠的 DEGs。通过纤维化生物标志物分离和鉴定 OFs。CP 和 CTSC 在 GO 组织样本和 TGF-β 刺激的 OFs 中下调。通过转染 CP 过表达载体或针对 CP 的小干扰 RNA（si1-CP 或 si2-CP）在 OFs 中实现 CP 过表达或敲低，并通过 qRT-PCR 验证。CP 过表达抑制细胞活力，降低α-SMA、波形蛋白、纤连蛋白和胶原 I 的水平，而 CP 敲低对 OFs 产生相反的影响。CP 过表达抑制 Smad3、Erk1/2、p38、JNK 和 AKT 的磷酸化；相反，CP 敲低对上述因子的磷酸化产生相反的影响。
CP 在 GO 中下调，并抑制 GO 和正常 OFs 中纤维化相关蛋白的表达。CP 可能成为 GO 治疗方案中的一种有前途的治疗药物。
