Isoquercitrin protects HUVECs against high glucose-induced apoptosis through regulating p53 proteasomal degradation
Isoquercitrin protects HUVECs against high glucose-induced apoptosis through regulating p53 proteasomal degradation
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DOI：
10.3892/ijmm.2021.4955
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发表时间：
2021-07-01
影响因子：
5.4
通讯作者：
Tang, Qizhu
中科院分区：
文献类型：
--
作者：
Liu, Libo;Huang, Sihui;Tang, Qizhu
高糖诱导的内皮细胞凋亡在糖尿病血管功能障碍中起重要作用。异槲皮苷（IQC）是一种黄酮苷类化合物，具有抗糖尿病作用，但其作用机制有待进一步研究。本研究观察了IQC对汞诱导的人脐静脉内皮细胞（HUVECs）凋亡的影响，并探讨其分子机制。HUVEC用5或30 mM葡萄糖处理48 h。使用细胞计数试剂盒-8测定法监测内皮细胞活力。JC-1染色检测线粒体膜电位。通过原位末端标记（TUNEL）染色和流式细胞术观察细胞凋亡。Western印迹法用于分析凋亡相关蛋白Bax、Bcl-2、切割的（C）-caspase 3、总-caspase 3、p53和磷酸化的p53。逆转录-定量PCR检测Bax、Bcl-2和p53 mRNA表达水平。免疫荧光染色检测HUVECs中p53和泛素特异性肽酶10（USP 10）的表达水平和分布。结果显示，IQC显著减弱HG诱导的内皮细胞凋亡，如TUNEL、JC-1染色和流式细胞术观察到的凋亡细胞减少所示。此外，IQC处理显著抑制HG应激下HUVECs中促凋亡蛋白p53、Bax和C-caspase 3的表达水平，并增加抗凋亡蛋白Bcl-2的表达水平。然而，IQC对HG的抗凋亡作用在体外通过增加p53蛋白水平而部分减弱。IQC影响高糖诱导的Bax和Bcl-2 mRNA表达水平，但不影响p53的转录。值得注意的是，IQC抑制HG诱导的p53在Ser 15的磷酸化和USP 10的核转运，使p53不稳定并增加p53蛋白的蛋白酶体降解。目前的研究结果表明，IQC发挥了保护作用，对汞诱导的内皮细胞凋亡，通过调节蛋白酶体降解的p53蛋白，这表明IQC可作为一种新的治疗化合物，以改善DM诱导的血管并发症。
High glucose (HG)-induced endothelial apoptosis serves an important role in the vascular dysfunction associated with diabetes mellitus (DM). It has been reported that isoquercitrin (IQC), a flavonoid glucoside, possesses an anti-DM effect, but the mechanism requires further investigation. The present study investigated the effect of IQC against HG-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and explored its molecular mechanism. HUVECs were treated with 5 or 30 mM glucose for 48 h. Endothelial cell viability was monitored using the Cell Counting Kit-8 assay. Mitochondrial membrane potential was detected by JC-1 staining. Apoptosis was observed by TUNEL staining and flow cytometry. Western blotting was used for the analysis of apoptosis-associated proteins Bax, Bcl-2, cleaved (C)-caspase3, total-caspase3, p53 and phosphorylated p53. Reverse transcription-quantitative PCR was used to analyze the mRNA expression levels of Bax, Bcl-2 and p53. Immunofluorescence staining was utilized to detect the expression levels and distribution of p53 and ubiquitin specific peptidase 10 (USP10) in HUVECs. The results revealed that IQC significantly attenuated HG-induced endothelial apoptosis, as shown by decreased apoptotic cells observed by TUNEL, JC-1 staining and flow cytometry. Moreover, under HG stress, IQC treatment markedly inhibited the increased expression levels of the pro-apoptotic proteins p53, Bax and C-caspase3, and increased the expression levels of the anti-apoptotic protein Bcl-2 in HUVECs. However, the anti-apoptotic effect of IQC against HG was partially blunted by increasing p53 protein levels in vitro. IQC influenced the mRNA expression levels of Bax and Bcl-2 in response to HG, but it did not affect the transcription of p53. Notably, IQC inhibited the HG-induced phosphorylation of p53 at Ser15 and the nuclear transport of USP10, destabilizing p53 and increasing the proteasomal degradation of the p53 protein. The current findings revealed that IQC exerted a protective effect against the HG-induced apoptosis of endothelial cells by regulating the proteasomal degradation of the p53 protein, suggesting that IQC may be used as a novel therapeutic compound to ameliorate DM-induced vascular complications.