■Notch信号传导对于调节血管内皮细胞(EC)的功能至关重要。然而,Notch1胞内结构域(NICD)对脓毒症EC损伤的影响尚不清楚.
■我们通过脂多糖(LPS)注射和盲肠结扎穿孔(CLP)在小鼠模型中建立了血管内皮功能障碍和诱发脓毒症的细胞模型。使用CCK-8,通透性,流式细胞术,免疫印迹,和免疫沉淀测定。体外评价NICD抑制或活化对内皮屏障功能的影响。褪黑素用于脓毒症小鼠的NICD激活。存活率,器官的伊文思蓝染料,血管松弛测定,免疫组织化学,ELISA,采用免疫印迹法探讨褪黑素在脓毒症血管功能障碍中的作用。
■我们发现LPS,白细胞介素6和脓毒症患儿血清可抑制NICD及其下游调节因子Hes1的表达,从而损害内皮屏障功能并通过AKT途径导致EC凋亡。机械上,LPS通过抑制去泛素化酶的表达降低NICD的稳定性,泛素特异性蛋白酶8(USP8)。褪黑激素,然而,USP8表达上调,从而保持NICD和Notch信号的稳定性,这最终减少了我们的脓毒症模型中的EC损伤并提高了脓毒症小鼠的存活率。
■我们发现,在脓毒症期间,Notch1在介导血管通透性方面的作用,我们发现抑制NICD会导致脓毒症患者的血管EC功能障碍,它被褪黑激素逆转了。因此,Notch1信号通路是治疗脓毒症的潜在靶点。
Notch signaling is critical for regulating the function of vascular endothelial cells (ECs). However, the effect of the intracellular domain of Notch1 (
NICD) on EC injury in sepsis remains unclear.
We established a cell model of vascular endothelial dysfunction and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Endothelial barrier function and expression of endothelial-related proteins were determined using CCK-8, permeability, flow cytometry, immunoblot, and immunoprecipitation assays. The effect of
NICD inhibition or activation on endothelial barrier function was evaluated in vitro. Melatonin was used for
NICD activation in sepsis mice. The survival rate, Evans blue dye of organs, vessel relaxation assay, immunohistochemistry, ELISA, immunoblot were used to explore the specific role of melatonin for sepsis induced vascular dysfunction in vivo.
We found that LPS, interleukin 6, and serum collected from septic children could inhibit the expression of
NICD and its downstream regulator Hes1, which impaired endothelial barrier function and led to EC apoptosis through the AKT pathway. Mechanistically, LPS decreased the stability of
NICD by inhibiting the expression of a deubiquitylating enzyme, ubiquitin-specific proteases 8 (USP8). Melatonin, however, upregulated USP8 expression, thus maintaining the stability of NICD and Notch signaling, which ultimately reduced EC injury in our sepsis model and elevated the survival rate of septic mice.
We found a previously uncharacterized role of Notch1 in mediating vascular permeability during sepsis, and we showed that inhibition of NICD resulted in vascular EC dysfunction in sepsis, which was reversed by melatonin. Thus, the Notch1 signaling pathway is a potential target for the treatment of sepsis.