ORM1 Effect on Sphingolipid Metabolism in Dry Eye via SPTLC1
-
摘要:
目的 研究人源血清类粘蛋白1（orosomucoid 1，ORM1）通过丝氨酸棕榈酰转移酶长链碱基亚基 1（serine palmitoyltransferase long chain base subunit 1，SPTLC1）对干眼症鞘脂代谢的影响，为干眼症的发病机制提供新的研究方向。 方法 通过皮下注射氢溴酸东莨菪碱构建干眼症模型，通过晶状体注射腺相关病毒过表达ORM1。行泪液分泌量检测，杯状细胞检测，角膜荧光素染色，泪膜破裂时间检测，HE检测评估角膜结膜损伤，泪液质量，以及组织病理变化。通过生化试剂盒检测神经酰胺和鞘磷脂含量，通过qPCR和WB检测ORM1和SPTLC1的表达。 结果 （1） ORM1 可增加干眼症模型的泪液分泌（P < 0.0001 ）；（2）ORM1 可增加干眼症模型的眼表泪膜稳定性（P < 0.001）；（3）ORM1 可改善干眼症模型的角膜上皮损伤（P <0.0001 ）；（4）ORM1 可增加干眼症模型的角膜组织中杯状细胞数量（P < 0.001）；（5）过表达ORM1后，角膜组织上皮层变厚，基底层细胞排列较为紧密，细胞层数变多，空泡减少；（6）ORM1 可抑制干眼症模型的炎症基因表达（P < 0.01）；（7）ORM1 可促进总神经酰胺和鞘磷脂含量（P < 0.01）；（8）ORM1 可促进SPTLC1的mRNA和蛋白表达（P < 0.001）。结论 ORM1可通过调控SPTLC1参与调控干眼症鞘脂代谢，为探讨干眼症的发生发展机制和寻找有效且安全的治疗方案提供新的视角。 Abstract:Objective To study the effect of ORM1 on sphingolipid metabolism in dry eye via SPTLC1 so as to provide a new research direction for the pathogenesis of dry eye. Methods By the subcutaneous injection of scopolamine hydrobromide （SCOP）, a dry eye model was constructed, and adeno-associated virus overexpressing ORM1 was injected through the lens. Tear secretion assay, goblet number, corneal fluorescein staining, lacrimal gland rupture time, and HE assay were performed to assess the corneal conjunctival damage, tear quality, and histopathological changes. Ceramide and sphingomyelin content were detected by biochemical kits, and the expression of ORM1 and SPTLC1 was detected by qPCR and WB. Results 1. ORM1 increased tear secretion in the dry eye model （P < 0.0001 ）; 2. ORM1 increased the stability of the lacrimal gland on the ocular surface in the dry eye model （P <0.0001 ）; 3. ORM1 improved the corneal epithelial damage in the dry eye model （P <0.0001 ）; 4. ORM1 increased the number of goblet in the corneal tissue in the dry eye model （P <0.0001 ）; 5. After the overexpression of ORM1, the epithelium of the corneal tissue became thicker and the cells of the basal lamina were more closely arranged. cell layers became more numerous and vacuoles were reduced; 6. ORM1 inhibited the inflammatory gene expression in a dry eye model （P < 0.01）; 7. ORM1 promoted the total ceramide and sphingomyelin content （P < 0.01）; 8. ORM1 promoted the rise of mRNA and protein expression of SPTLC1 （P < 0.001）.Conclusion ORM1 can participate in the regulation of sphingolipid metabolism in dry eye disease through the regulation of SPTLC1, which provides new perspectives for exploring the mechanism of the development of dry eye disease and searching for effective and safe therapeutic options. -
Key words:
- ORM1 /
- SPTLC1 /
- Sphingolipid metabolism /
- Dry eye disease
-
图 1 ORM1 过表达促进干眼症模型的泪液分泌量
A： qPCR检测ORM1的mRNA表达；B：Western blot 检测ORM1的蛋白表达C：各组酚红棉线长度；D：泪液分泌量；**P < 0.01，****P < 0.0001，####P < 0.0001。
Figure 1. ORM1 overexpression promotes tear production in a dry eye model
图 3 ORM1 过表达能够降低小鼠角膜荧光素染色评分
A：角膜荧光素染色；B：角膜荧光素染色得分；****P < 0.0001。
Figure 3. ORM1 overexpression reduces mouse corneal fluorescein staining score
图 4 ORM1 过表达能够增多小鼠角膜组织中杯状细胞数量
A：通过PAS染色评估结膜杯状细胞数量 （x40）; B: 角膜杯状细胞数量;***P < 0.001，***P < 0.0001。
Figure 4. ORM1 overexpression increases the number of cup cells in mouse corneal tissue
图 5 ORM1 过表达能够改善小鼠病理变化
Figure 5. ORM1 overexpression improves pathological changes in mice
图 6 通过qPCR检测角膜组织中炎症基因IL-1β，IL-6，TNF-α 和IFN- γ的表达
A：IL-1β的表达; B: IL-6的表达; C: TNF-α的表达; D: IFN- γ的表达；**P < 0.01，****P < 0.0001。
Figure 6. Expression of inflammatory genes IL-1β，IL-6，TNF-α and IFN- γ in corneal tissues detected by qPCR
图 7 ORM1 过表达促进总神经酰胺和鞘磷脂含量
A: 总神经酰胺含量;B:鞘磷脂含量; **P < 0.01，*** P < 0.001，****P < 0.0001。
Figure 7. ORM1 overexpression promotes total ceramide and sphingomyelin content
图 8 ORM1 过表达调控SPTLC1表达
A: 通过string预测与ORM1互作的蛋白;B: 通过Human protein atlas 对SPTLC1在眼单细胞的表达进行预测; C: 通过Human protein atlas 对SPTLC3在眼单细胞的表达进行预测; D: qPCR检测小鼠的角膜组织中的SPTLC1的mRNA表达; E: 小鼠的角膜组织中的SPTLC1的蛋白表达; *P < 0.05， ** P < 0.01，***P < 0.001，**** P < 0.0001。
Figure 8. ORM1 overexpression regulates SPTLC1 expression
表 1 荧光定量PCR扩增引物列表
Table 1. Nucleotide sequences of the primers used for real-time quantitative PCR
引物名称 引物序列 长度（bp） IL-1β forward 5′-GGGGCGTCCTTCATATGTGT-3′ 347 IL-1β reverse 5′-GGCAGCTCCTGTCTTGTAGG-3′ IL-6 forward 5′-GAGGTGAGTGCTTCCCCATC-3′ 477 IL-6 reverse 5′-TTGCATCTGGCTTTGTTCGC-3′ TNF-α forward 5′-ACTGATGAGAGGGAGGCCAT -3′ 179 TNF-α reverse 5′-CCGTGGGTTGGACAGATGAA-3′ IFN- γ forward 5′-ATCAAGCTGCCTCCCGTATG-3′ 942 IFN- γ reverse 5′-CTGTCTGCAGTGGGGAAACA-3′ ORM1 forward 5′-GGACACCTGTGGGCTATGTC-3′ 281 ORM1 reverse 5′-TGACCGCACCTATCCTGGAA-3′ SPTLC1 forward 5′-CAGTGCTGCACAGAAACCTC-3′ 603 SPTLC1 reverse 5′-TTGTCTTGCTCTTCTCCCTCAC-3′ β-actin forward 5′-CCTAAGGCCAACCGTGAAAAG -3′ 100 β-actin reverse 5′-AGGCATACAGGGACAGCACAG-3′ 
下载: 导出CSV
表 2 Western blot中所用一抗
Table 2. Primary antibodies used in western blot
一抗 公司 货号 稀释比 分子量 种属 ORM1 abcam ab200732 1∶ 1000 24 Rabbit SPTLC1 abcam Ab176706 1∶ 2000 53 Rabbit β-actin abcam Ab8226 1∶ 1000 42 Mouse
下载: 导出CSV
-
[1] Sheppard J,Shen Lee B,Periman L M. Dry eye disease: Identification and therapeutic strategies for primary care clinicians and clinical specialists[J]. Ann Med,2023,55(1):241-252. doi: 10.1080/07853890.2022.2157477 [2] Huang R,Su C,Fang L,et al. Dry eye syndrome: Comprehensive etiologies and recent clinical trials[J]. Int Ophthalmol,2022,42(10):3253-3272. doi: 10.1007/s10792-022-02320-7 [3] 金明,王晓娟,宋海姣,等. 中药及熏灸治疗干眼症的临床观察[J]. 中国中医眼科杂志,2006,16(2):71-73. [4] Rolando M,Barabino S. Dry eye disease: What is the role of vitamin D?[J]. Int J Mol Sci,2023,24(2):1458. doi: 10.3390/ijms24021458 [5] 王少媛,张金兰,张丹,等. 鞘脂在肝脏疾病中的研究进展[J]. 药学学报,2015,50(12):8. [6] Mei M,Liu M,Mei Y,et al. Sphingolipid metabolism in brain insulin resistance and neurological diseases[J]. Front Endocrinol (Lausanne),2023,14(1):1243132. [7] 王静,胡小松,石阶平. 鞘脂与细胞凋亡[J]. 生理科学进展,2003,34(3):5. [8] Haslam T M,Feussner I. Diversity in sphingolipid metabolism across land plants[J]. J Exp Bot,2022,73(9):2785-2798. doi: 10.1093/jxb/erab558 [9] 钱益勇,李侲宇,张振平,等. 鞘脂酶激活蛋白在年龄相关性白内障晶状体上皮细胞的表达[J]. 眼视光学杂志,2007,9(3):4. [10] 黄江丽,张抒燕,辛瑞. 加味沙参麦冬汤+中药熏蒸治疗干眼症的效果及角膜荧光素染色评分分析[J]. 中外医疗,2024,(9). [11] 赵凤琼,李立. 依达拉奉对糖尿病角膜神经病变大鼠角膜神经的保护作用[J]. 中华实验眼科杂志,2012,30(2):5. [12] 范围. 糖尿病角膜神经病变与视网膜病变的相关性研究[D]. 重庆: 中国人民解放军陆军军医大学/第三军医大学,2016. [13] 金梅,罗晓燕,曲利利. 19例合并2型糖尿病的干眼症患者干眼症症状,角膜神经病变观察及相关性分析[J]. 山东医药,2022,62(12):64-67. [14] 孙九丽,林慧珍,苟萍. 鞘脂代谢及其相关疾病研究进展[J]. 生物技术,2011,21(5):5. [15] 王睿齐,张庆柱. 肿瘤鞘脂代谢异常与肿瘤细胞对维甲酸类药物耐药性[J]. 生命的化学,2014,34(6):6. [16] Sasset L,Di Lorenzo A. Sphingolipid metabolism and signaling in endothelial cell functions[J]. Adv Exp Med Biol,2022,1372(1):87-117. [17] Kalinichenko L S,Gulbins E,Kornhuber J,Müller C P. Sphingolipid control of cognitive functions in health and disease[J]. Prog Lipid Res,2022,86:101162. doi: 10.1016/j.plipres.2022.101162 [18] Gemelli C,Martello A,Montanari M,et al. The Orosomucoid 1 protein is involved in the vitamin D - mediated macrophage de-activation process[J]. Exp Cell Res,2013,319(20):3201-3213. doi: 10.1016/j.yexcr.2013.08.017 [19] Kim Y E,Lee E J,Kim K,et al. Urine SERPINC1/ORM1 as biomarkers for early detection of lupus nephritis in MRL-lpr mice[J]. Front Immunol,2023,14:1148574. doi: 10.3389/fimmu.2023.1148574 [20] Yu G,Gao J,Hu W,et al. ORM1 promotes tumor progression of kidney renal clear cell carcinoma (KIRC) through CALR-mediated apoptosis[J]. Sci Rep,2023,13(1):15687. doi: 10.1038/s41598-023-42962-w [21] Yang B F,Zhai F,Yu S,et al. Evaluation of IFIT3 and ORM1 as biomarkers for discriminating active tuberculosis from latent infection[J]. Curr Med Sci,2022,42(6):1201-1212. doi: 10.1007/s11596-022-2649-6 [22] Master A,Kontzias A,Huang L,et al. The transcriptome of rabbit conjunctiva in dry eye disease: Large-scale changes and similarity to the human dry eye[J]. PLoS One,2021,16(7):e0254036. doi: 10.1371/journal.pone.0254036 [23] Schäfer J H,Körner C,Esch B M,et al. Structure of the ceramide-bound SPOTS complex[J]. Nat Commun,2023,14(1):6196. doi: 10.1038/s41467-023-41747-z [24] 郑雪莹. 鞘脂代谢酶SPTLC1促进胃癌细胞增殖及其抑制有丝分裂灾难的机制研究[D]. 北京: 中国医科大学,2022. -
点击查看大图
计量
- 文章访问数: 55
- HTML全文浏览量: 89
- PDF下载量: 8
- 被引次数: 0
