香茅醛通过激活自噬-溶酶体途径促进RNF4降解以拮抗内皮细胞氧化损伤

Citronellal antagonizes oxidative damage in endothelial cells by activating the autophagylysosome pathway to promote the degradation of RNF4

摘要:

目的]探讨香茅醛是否通过增强自噬-溶酶体途径,促进环指蛋白4(RNF4)降解,进而稳定鸟苷三磷酸环化水解酶1(GTPCH1)的蛋白水平,以拮抗氧化应激诱导的人脐静脉内皮细胞损伤。 [方法]采用H₂O₂处理人脐静脉内皮细胞构建氧化损伤模型,并用0.48 mmol/L香茅醛进行干预。MTT测定细胞活力,Western blot检测p62、微管相关蛋白轻链3B(LC3B)、溶酶体相关膜蛋白1(LAMP1)、GTPCH1和RNF4蛋白表达。使用溶酶体抑制剂氯喹(CQ)和蛋白酶体抑制剂MG132处理细胞。免疫共沉淀(CoIP)验证在H₂O₂作用下GTPCH1的SUMO化及其与RNF4的相互作用。 [结果]与对照组相比,H₂O₂处理可显著降低内皮细胞活力(P＜0.05)及GTPCH1蛋白水平(P＜0.01),并上调RNF4蛋白表达(P＜0.01)；CoIP实验证实,H₂O₂可诱导GTPCH1发生SUMO化修饰并与RNF4结合。与H₂O₂组相比,香茅醛干预可显著逆转上述变化,同时改善自噬流功能:p62水平显著降低(P＜0.001),LC3B-Ⅱ/LC3B-Ⅰ比值恢复正常(P＜0.001),LAMP1表达上调(P＜0.05)。抑制剂实验显示,与H₂O₂组相比,MG132可显著阻断GTPCH1的降解(P＜0.01),CQ可显著抑制RNF4的降解(P＜0.05)。 [结论]H₂O₂通过诱导RNF4介导的GTPCH1 SUMO化-泛素化降解引发内皮细胞损伤；香茅醛通过增强自噬-溶酶体通路功能促进RNF4降解,进而稳定GTPCH1,拮抗H₂O₂所致的内皮细胞损伤。

Abstract:

Aim To investigate whether citronellal antagonizes oxidative stress-induced injury in human umbilical vein endothelial cells by enhancing the autophagy-lysosome pathway, promoting the degradation of ring finger protein 4 (RNF4) and thereby stabilizing the protein levels of guanosine triphosphate cyclohydrolase 1 (GTPCH1). Methods Human umbilical vein endothelial cells were treated with H₂O₂ to establish oxidative damage models, and then treated with 0.48 mmol/L citronellal. Cell viability was determined by MTT assay; the protein expressions of p62, microtubule-associated protein light chain 3B (LC3B), lysosome-associated membrane protein 1 (LAMP1), GTPCH1 and RNF4 were detected by Western blot. The cells were treated with the autophagy inhibitor chloroquine (CQ) and the proteasome inhibitor MG132. Co-immunoprecipitation (CoIP) was used to verify the GTPCH1 SUMOylation and its interaction with RNF4 in response to H₂O₂. Results Compared with the control group, H₂O₂ treatment significantly decreased endothelial cell viability (P＜0.05) and GTPCH1 protein level (P＜0.01) and up-regulated RNF4 protein expression (P＜0.01). CoIP assay confirmed that H₂O₂ induced GTPCH1 to undergo SUMOylation and bind to RNF4. Compared with the H₂O₂ group, citronellal treatment significantly reversed the above changes and improved the autophagic flux:p62 was significantly decreased (P＜0.001); the LC3B-Ⅱ/LC3B-Ⅰ ratio was restored (P＜0.001); and the expression of LAMP1 was up-regulated (P＜0.05). Inhibitor experiments showed that MG132 blocked GTPCH1 degradation (P＜0.01) and CQ inhibited RNF4 degradation (P＜0.05) compared with the H₂O₂ group. Conclusion H₂O₂ triggers endothelial cell injury via RNF4-mediated GTPCH1 SUMOylation-ubiquitination degradation; citronellal promotes the degradation of RNF4 by enhancing the function of the autophagy-lysosome pathway, thereby stabilizing GTPCH1 and antagonizing H₂O₂-induced endothelial cell injury.