目的：探讨糖尿病氧化应激环境中过表达α-Klotho(KL)的小鼠单核巨噬细胞白血病细胞(RAW264.7)对人脐静脉内皮细胞(HUVECs)增生、迁移、管腔形成以及紧密连接的影响。

方法：将RAW264.7细胞分为对照组、4-羟壬二酸酯(4HNE)组、4HNE+KL组，采用免疫荧光实验检测RAW264.7细胞F4/80的表达。制备3组细胞的条件培养基用于培养HUVECs，分为Mø；-NC组、Mø；-4HNE组和Mø；-4HNE+KL组。采用CCK8实验检测血管内皮细胞增生，采用划痕实验和Transwell实验检测迁移，采用管腔形成实验检测管腔形成，采用Western blot实验检测闭合蛋白5(Claudin 5)、咬合蛋白(Occludin)、带状闭合蛋白1(ZO 1)表达水平。

结果：免疫荧光实验结果显示，4HNE组RAW264.7细胞F4/80荧光强度较对照组明显增强，而4HNE+KL组F4/80荧光强度较4HNE组明显减弱(均P<0.05)。CCK8实验结果显示，相比于Mø；-NC组，Mø；-4HNE组HUVECs增生显著增加，而Mø；-4HNE+KL组HUVECs增生较Mø；-4HNE组显著下降(均P<0.01)。划痕实验和Transwell实验结果显示，相比于Mø；-NC组，Mø；-4HNE组HUVECs迁移显著增强，而Mø；-4HNE+KL组HUVECs迁移较Mø；-4HNE组显著减弱(均P<0.01)。管腔形成实验结果显示，相比于Mø；-NC组，Mø；-4HNE组HUVECs管腔数显著增加，而Mø；-4HNE+KL组管腔数较Mø；-4HNE组显著下降(均P<0.01)。Western blot实验结果显示，相比于 Mø；-NC组，Mø；-4HNE组HUVECs中Claudin 5、Occludin、ZO 1蛋白的相对表达量明显减少，而Mø；-4HNE+KL组Claudin 5、Occludin、ZO 1蛋白的相对表达量较Mø；-4HNE组明显增加(均P<0.01)。

结论：KL通过改变糖尿病氧化应激环境中巨噬细胞激活状态抑制了HUVECs的增生、迁移、管腔形成，并增强了HUVECs的紧密连接。

METHODS:RAW264.7 cells were categorized into control, 4-hydroxynonenal(4HNE), and 4HNE+KL groups, with F4/80 expression assessed via immunofluorescence staining. Three groups of conditional media were prepared for HUVECs and culture divided into Mø-NC, Mø-4HNE, and Mø-4HNE+KL groups. Cell proliferation was evaluated using CCK8 assay, while scratch test and Transwell assays were employed to measure cell migration. Additionally, tube-formation assay was conducted to assess cell tubule formation, and Western blot assay was utilized to detect the protein expression levels of Claudin 5, Occludin and ZO 1.

RESULTS:The results of immunofluorescence staining showed that the fluorescence intensity of F4/80 of RAW264.7 cells in the 4HNE group was significantly enhanced compared with the control group, while that of F4/80 in the 4HNE+KL group was significantly decreased compared with the 4HNE group(all P<0.05). The CCK8 assay results revealed a significant increase in the proliferation of HUVECs in the Mø-4HNE group compared with the Mø-NC group. Conversely, the proliferation of the Mø-4HNE+KL group exhibited a significant decrease compared with that in the Mø-4HNE group(all P<0.01). The results of scratch test and Transwell assays demonstrated a significant increase in the migration of HUVECs in the Mø-4HNE group compared with the Mø-NC group, while the migration of the Mø-4HNE+KL group exhibited a significant decrease compared with the Mø-4HNE group(all P<0.01). In the tube-formation assay, it was observed that the number of tubes formed by HUVECs in the Mø-4HNE group was significantly increased compared with the Mø-NC group, while that of tubes formed in the Mø-4HNE+KL group was significantly decreased compared with the Mø-4HNE group(all P<0.01). Additionally, the Western blot results revealed a significant decrease in the relative expression levels of Claudin 5, Occludin, and ZO 1 in the Mø-4HNE group compared with the Mø-NC group. Conversely, in the Mø-4HNE+KL group, there was a significant increase in the relative expression levels of Claudin 5, Occludin, and ZO 1 compared to the Mø-4HNE group(all P<0.01).

CONCLUSIONS: KL inhibits the proliferation, migration, and tube-formation of HUVECs while enhancing the tight junction by changing the activation state of macrophages in the diabetic oxidative stress environment.