Objective: To investigate the role of p38MAPK signaling pathway in autophagy of intestinal epithelial cells induced by spvB of S.typhimurium.
Methods: Henle-407 cells in exponential growth were infected with wild-type S.typhimurium strain STM-211 (with spvB gene), spvB mutated strain STM-delata;spvB, or with delata;spvB-complemented strain STM-c-spvB after treatment of the cells with the p38MAPK inhibitor SB203580. At different time points of co-culture, the cells were collected and the intracellular bacteria were counted. Western blotting was performed to detect the expressions of phosphorylated p38 and autophagy-related proteins LC3 and p62; immunofluorescence staining was used to observe the expression and distribution of LC3.
Results: At 1, 2 and 4 h after the infection, the phosphorylation levels of p38 in STM-211 group and STM-c-spvB group were significantly lower than that in STM-delata;spvB group (P<0.05). At 2 and 4 h of co-culture, the intracellular bacterial counts were significantly greater in STM-211 and STM-c-spvB infection groups than in STM-delata;spvB group (P<0.05). Pretreatment with p38 inhibitor SB203580 did no significantly affect the expression levels of LC3 II or P62 in STM-211 and STM-c-spvB groups, but caused significant reduction in their expressions in STM-delata;spvB group at 1 h (P<0.05), and such changes were more obvious at 3 h (P<0.05).
Conclusion: The inhibitory effect of spvB gene on autophagy in intestinal epithelial cells is related with the negative regulation of p38MAPK signaling pathway.
目的: 探讨p38MAPK信号通路在鼠伤寒沙门菌spvB基因影响肠上皮细胞自噬中的作用。
方法: 分别使用携带spvB基因的鼠伤寒沙门氏菌野生株STM-211、敲除spvB基因的鼠伤寒沙门氏菌突变株STM-ΔspvB和回补株STM-c-spvB与处于生长对数期的肠黏膜上皮细胞Henle-407细胞进行共培养，并加入p38MAPK通路抑制剂SB203580进行干预。于共培养后的不同时间点收集细胞，通过免疫印迹法检测p38的磷酸化水平和自噬标志蛋白LC3、P62的表达水平；稀释涂板作胞内细菌计数；免疫荧光染色观察自噬蛋白LC3的表达及分布。
结果: 共培养后1、2、4 h，STM-211组和STM-c-spvB组p38蛋白的磷酸化水平均低于STM-ΔspvB组（P < 0.05）；在共培养后的2 h和4 h，STM-211组和STM-c-spvB组胞内活菌计数明显多于STM-ΔspvB组（P < 0.05）；在1 h时间点使用SB203580干预后，STM-211组和STM-c-spvB组LC3Ⅱ和P62表达量未见明显改变，而STM-ΔspvB组LC3Ⅱ表达量降低（P < 0.05），P62表达量升高（P < 0.05），3 h时该趋势更加明显（P < 0.05）。
结论: 鼠伤寒沙门氏菌spvB基因对肠上皮细胞自噬的抑制作用，可能与其对细胞p38MAPK通路的负调控相关。