探讨锯齿状经典缺刻配体2(jagged canonical Notch ligand 2, Jag2)通过肾血管内皮细胞-单核巨噬细胞互作介导急性肾损伤早期炎症的机制。

采用雄性C57BL/6J小鼠建立双侧肾缺血再灌注损伤(ischemia-reperfusion injury，IRI)模型，将小鼠随机分为假手术组、模型组(IRI建模前24 h和建模后即刻给予尾静脉注射10 mg/kg同型对照IgG)和Jag2阻断组(IRI建模前24 h和建模后即刻给予尾静脉注射等量的Jag2中和抗体)，每组各6只。利用组织病理染色、测定血清肌酐及血尿素氮和Western印迹法等评估肾损伤和蛋白表达。以人脐静脉血管内皮细胞(human umbilical vein endothelial cell，HUVEC)构建缺氧复氧模型，分别将缺氧复氧后的对照组HUVEC和敲低Jag2基因组HUVEC与人单核细胞白血病细胞株(tohoku hospital pediatrics-1，THP-1)进行共培养，然后以流式细胞术检测比较组间CD86^＋细胞比例。使用外源性Jag2重组蛋白刺激小鼠单核巨噬细胞白血病细胞株Raw264.7后，通过转录组测序筛选相关信号通路变化，并以Western印迹、实时荧光定量聚合酶链反应进行验证。

IRI建模后小鼠肾皮髓交界区的肾小管损伤显著，血清肌酐和血尿素氮升高，肾损伤分子-1、白细胞介素-1β表达显著上调(P均<0.05)；免疫荧光显示Jag2与内皮细胞标志物内皮黏蛋白共定位，且模型组的肾组织Jag2荧光阳性面积明显大于假手术组；干预实验表明，Jag2中和抗体显著减轻IRI小鼠的肾小管坏死，减轻肾功能恶化，并下调肾损伤分子-1和白细胞介素-1β表达(P均<0.05)。缺氧复氧处理后的共培养实验表明，敲除Jag2组的HUVEC与THP-1细胞共培养后，CD86^＋细胞比例显著低于对照组(P<0.05)；转录组测序显示，Jag2激活了磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase，PI3K)-蛋白激酶B(protein kinase B，AKT)信号通路等多种炎症相关信号通路。Raw264.7细胞实验也证明，Jag2能够上调小鼠单核巨噬细胞的磷酸化AKT蛋白水平，以及白细胞介素-1β和白细胞介素-6的mRNA水平(P均<0.05)。

Jag2能够通过肾血管内皮细胞-单核巨噬细胞互作诱导单核巨噬细胞向促炎表型转化、激活PI3K-AKT等炎症相关信号通路、促进炎症因子产生，导致急性肾损伤早期炎症进展。

To investigate the mechanism of jagged canonical Notch ligand 2 (Jag2) mediating early inflammation in acute kidney injury via crosstalk between renal vascular endothelial cells and mononuclear macrophages.

Male C57BL/6J mice were used to establish a bilateral renal ischemia-reperfusion injury (IRI) model. The mice were randomly divided into three groups with 6 mice in each group: the sham operation group, the model group (administered with 10 mg/kg isotype control IgG via tail vein injection at 24 h before IRI modeling and immediately after modeling), and the Jag2 blockade group (administered with an equal dose of Jag2 neutralizing antibody via tail vein injection at 24 h before IRI modeling and immediately after modeling). Renal injury and protein expression were assessed using histopathological staining, measurement of serum creatinine and urea nitrogen, and western blotting. Human umbilical vein endothelial cells (HUVEC) were used to establish a hypoxia/reoxygenation (H/R) model. After the H/R treatment, HUVEC in control group and Jag2 gene-knockdown group were co-cultured with human monocytic leukemia cell line tohoku hospital pediatrics-1 (THP-1), respectively. Then flow cytometry was applied to compare the proportion of CD86⁺ cells among the groups. After stimulating the mouse monocyte-macrophage leukemia cell line Raw264.7 with exogenous recombinant Jag2 protein, transcriptome sequencing was used to screen for changes in related signaling pathways, and the results were verified by Western blotting and qPCR.

After the renal IRI modeling, renal tubular injury at the corticomedullary junction of the mice kidneys was significant, with elevated levels of serum creatinine and blood urea nitrogen. The expressions of kidney injury molecule-1 and interleukin-1β were markedly upregulated (all P<0.05). Immunofluorescence results showed that Jag2 colocalized with the endothelial cell marker endomucin, and the fluorescence positive area of Jag2 in renal tissues of the model group was obviously larger than that in the sham operation group. Intervention experiments showed that Jag2 neutralizing antibody significantly alleviated the renal tubular necrosis, attenuated the deterioration of renal function, and downregulated the expression of kidney injury molecule-1 and interleukin-1β in the IRI mice (all P< 0.05). Co-culture experiments after the H/R treatment showed that the proportion of CD86⁺ cells was significantly lower in the HUVEC of Jag2 knockout group co-cultured with THP-1 cells than that in the HUVEC of the control group co-cultured with THP-1 cells (P<0.05). Transcriptome sequencing revealed that Jag2 activated multiple inflammation-related signaling pathways including the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Experiments on Raw264.7 cells also demonstrated that Jag2 could upregulate the protein level of phosphorylated AKT, as well as the mRNA levels of interleukin-1β and interleukin-6 in the mouse mononuclear macrophages (all P< 0.05).

Jag2 can induce the transformation of mononuclear macrophages into a pro-inflammatory phenotype through the crosstalk between renal vascular endothelial cells and mononuclear macrophages, activate inflammation-related signaling pathways such as PI3K-AKT, and promote the production of inflammatory cytokines, thereby driving the progression of inflammation in the early stage of acute kidney injury.