Zhao Yinhua, Lin Huang, Ma Jiaxing, Cui Yue
Plastic and Reconstructive Surgery, Beijing Anzhen Hospital, Capital Medical University, Chaoyang District, Beijing, 100029, China.
Heliyon. 2023 Jul 9;9(7):e17907. doi: 10.1016/j.heliyon.2023.e17907. eCollection 2023 Jul.
Botulinum toxin type A (BTXA) previously protected endothelial cells in free skin flaps from ischemia/reperfusion injury by inducing autophagy. Endoplasmic reticulum (ER) stress-autophagy activation may have a role in BTXA-antagonized ischemia/reperfusion damage in human dermal microvascular endothelial cells (HDMECs), however, this has yet to be proven. HDMECs were pretreated with BTXA at various concentrations for 12 h before being subjected to hypoxia and reoxygenation (H/R). Cell Count Kit 8 (CCK8) and Western blot (WB) data showed that H/R treatment significantly increased the expression of ER stress (GRP78, CHOP) and autophagy (LC3II/I, Beclin-1) proteins. The optimal BTXA pretreatment concentration was 1.6 U/mL, which resulted in the highest levels of cell survival and expression of ER stress and autophagy. Following pretreatment with 1.6 U/mL BTXA and the addition of the ER stress inducer Thapsigargin (Tg), the ER stress inhibitor 4-phenylbutyrate (4-PBA), and the inhibitor of autophagy Bafilomycin A1 (Baf A1), respectively, HDMECs were then subjected to H/R treatment. Cell survival and the percentage of ethynyldeoxyuridine-labeled cells in the BTXA pretreatment groups were reduced by the addition of Tg, 4-PBA, and Baf A1. While the expression of GRP78, CHOP, and LC3 was upregulated by Tg and Baf A1, it was downregulated by 4-PBA. The findings showed that ER stress produced by BTXA pretreatment triggers protective autophagy and protects HDMECs from H/R damage. There were no cytoprotective effects from either excessive activation or reduction of ER stress. Our results are consistent with the notion that autophagy and moderate ER stress are critical for cellular longevity and, consequently, functional integrity and may represent a potential therapeutic target.
A型肉毒杆菌毒素（BTXA）先前通过诱导自噬保护游离皮瓣中的内皮细胞免受缺血/再灌注损伤。内质网（ER）应激-自噬激活可能在BTXA拮抗的人真皮微血管内皮细胞（HDMECs）缺血/再灌注损伤中起作用，然而，这一点尚未得到证实。HDMECs在经历缺氧和复氧（H/R）之前，先用不同浓度的BTXA预处理12小时。细胞计数试剂盒8（CCK8）和蛋白质免疫印迹（WB）数据显示，H/R处理显著增加了内质网应激（GRP78、CHOP）和自噬（LC3II/I、Beclin-1）蛋白的表达。最佳的BTXA预处理浓度为1.6 U/mL，该浓度下细胞存活率以及内质网应激和自噬的表达水平最高。在用1.6 U/mL BTXA预处理并分别添加内质网应激诱导剂毒胡萝卜素（Tg）、内质网应激抑制剂4-苯基丁酸（4-PBA）和自噬抑制剂巴弗洛霉素A1（Baf A1）后，HDMECs再进行H/R处理。添加Tg、4-PBA和Baf A1后，BTXA预处理组中的细胞存活率和乙炔基脱氧尿苷标记细胞的百分比降低。虽然Tg和Baf A1上调了GRP78、CHOP和LC3的表达，但4-PBA使其下调。研究结果表明，BTXA预处理产生的内质网应激触发了保护性自噬，并保护HDMECs免受H/R损伤。内质网应激过度激活或降低均无细胞保护作用。我们的结果与自噬和适度的内质网应激对细胞寿命至关重要，进而对功能完整性至关重要的观点一致，并且可能代表一个潜在的治疗靶点。
