特异性Kv1.3阻滞剂调节巨噬细胞自噬在抗结肠炎中的作用

目的 探讨特异性Kv1.3阻滞剂PAP-1抗DSS结肠炎小鼠的作用机制。方法 将C57BL/6小鼠随机分为正常对照组、PAP-1对照组、DSS模型组、DSS+PAP-1组。5% DSS制备结肠炎模型,PAP-1(3 μg·g -1 ,每天3次,连续7 d)腹腔注射给药。进行DAI评分;实验结束行结肠HE染色评分,结肠组织匀浆检测MPO活性及炎症因子水平;免疫荧光显微镜观察结肠巨噬细胞改变;透射电镜观察巨噬细胞自噬结构;qPCR、Western blot检测小鼠结肠和巨噬细胞iNOS、IL-1β及自噬相关标志物表达。结果 PAP-1减少DSS结肠炎小鼠体质量的下降,降低DAI及结肠HI评分( P <0.05);PAP-1降低结肠MPO活性及促炎因子水平( P <0.05)。PAP-1降低结肠F4/80表达,使腹腔巨噬细胞及脾巨噬细胞内自噬泡增多;同时,PAP-1降低结肠、腹腔巨噬细胞和脾巨噬细胞iNOS、IL-1β及p62表达,升高LC3-Ⅱ及Beclin-1表达( P <0.05)。结论 PAP-1可能通过减少结肠炎小鼠巨噬细胞自噬受损,降低炎症因子水平,减轻结肠炎症损害。

The anti-colitic effect of specific Kv1.3 blocker in its regulation of macrophage autophagy

Aim To investigate the mechanism of the effect of a specific Kv1.3 blocker PAP-1 on DSS-induced colitis.Methods C57BL/6 mice were divided into normal control group, PAP-1 control group, DSS model group and DSS+PAP-1 group.DSS-induced colitis model was constructed by adding DSS (final concentration of 5%) to the drinking water of mice.PAP-1 (3 μg·g -1 ,3 times a day for 7 days) was administered intraperitoneally, while an equal volume of solvent was intraperitoneally injected to normal control group and model group.The changes of body weight of mice and the inflammatory activity index (DAI) were recorded every day.After the experiment, the peritoneal macrophages, spleen and colon tissues of mice were collected.Some colon tissues were used for colonic HE staining, and the tissue homogenate was used to detect MPO activities and inflammatory factors.Immunofluorescence confocal microscopy was used to observe the exudation of colonic macrophages.Transmission electron microscopy was used to observe the autophagy-related structures of macrophages, qPCR and Western blot were used to detect the expression of iNOS, IL-1β and autophagy- related markers LC3-Ⅱ, p62, and Beclin-1 in mouse colon, peritoneal macrophages and spleen macrophages.Results The DSS-induced mouse colitis model was successfully constructed.PAP-1 reduced weight loss of DSS-induced colitis mice ( P <0.05) and decreased DAI score ( P <0.05) and colon HI score ( P <0.05).The MPO activity and the levels of IL-1, IL-6 and TNF-α decreased in colon tissue of DSS+PAP-1 group compared to DSS model group ( P <0.05).The expression of F4/80 in DSS+PAP-1 group was significantly lower than that in DSS model group based on immunofluorescence microscopic observation.Under the electron microscope, the autophagic bubbles in peritoneal macrophages and spleen macrophages in DSS+PAP-1 group increased compared with those in DSS model group.qPCR and Western blot showed that in DSS+PAP-1 group, compared with DSS model group, the expression of iNOS, IL-1β and p62 significantly decreased, while the expression of LC3-Ⅱ and Beclin-1 markedly increased in colon, peritoneal macrophages and spleen macrophages ( P <0.05).Conclusions PAP-1 reduces the impaired autophagy of macrophages in mice with colitis, further decreases inflammatory cytokine production, and ultimately ameliorates the immunoinflammatory damage of colitis.