ORM1 Effect on Sphingolipid Metabolism in Dry Eye via SPTLC1
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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
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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 （×40）；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；F：各组小鼠角膜组织中SPLC1蛋白的相对表达量；*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′ 
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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
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