CD11b激动剂leukadherin-1对小鼠实验性结肠炎发病的影响及其机制研究

Effects of CD11b agonist leukadherin-1 on dextran sulfate sodium-induced acute experimental colitis in mice and the underlying mechanism

目的研究CD11b激动剂leukadherin-1(LA1)对小鼠实验性结肠炎模型肠道炎症发生发展的影响及其机制。方法构建硫酸葡聚糖钠盐(DSS)诱导的小鼠实验性结肠炎模型,检测小鼠体重变化,死亡情况和结肠长度,用HE染色评估结肠组织病理损伤,采用TUNEL法检测结肠组织中凋亡细胞的比例,采用比色法检测结肠组织髓过氧化物酶(MPO)活性,采用ELISA检测结肠IL-1β和TNF-α水平,并且采用免疫荧光共聚焦实验检测结肠组织巨噬细胞及TNF-α的水平。LA1处理小鼠0、3、6和12 h,采用流式细胞术检测小鼠体内巨噬细胞表面TLR4的表达变化。LA1处理骨髓来源巨噬细胞(BMDMs)0、3、6和12 h,采用蛋白免疫印迹技术检测Toll样受体4(Toll-like receptor 4,TLR4)的表达总量。采用非配对t检验进行统计学分析。结果与DSS组相比较,LA1+DSS处理组小鼠的死亡率显著降低,体重降低减缓,结肠较长,且差异有统计学意义。与DSS组相比较,LA1+DSS处理组的HE切片病理损伤明显减轻,细胞凋亡比例明显减少,MPO活性较低,结肠组织中巨噬细胞减少,IL-1β和TNF-α相对含量较低,且差异有统计学意义。与未处理的巨噬细胞相比,LA1处理的巨噬细胞中TLR4的表达总量无明显改变,而巨噬细胞表面TLR4平均荧光强度显著减弱。结论CD11b激动剂LA1通过抑制巨噬细胞TLR4通路活化缓解DSS诱导的小鼠实验性结肠炎,本研究为人们理解和探索炎症性肠病的发病机制提供了新思路和理论参考。

Objective To investigate the role of CD11b agonist leukadherin-1(LA1)in the development of intestinal inflammation and colitis disease in a mouse model of dextran sulfate sodium(DSS)-induced colitis.Methods The mouse model of experimental colitis was induced by DSS.Body weight changes and survival status were monitored every day.The length of colons was measured at day 7.Colon tissue sections were stained with hematoxylin and eosin(HE)and observed under an optical microscope for pathological analysis.The percentages of apoptotic cells in colon tissues were observed by TUNEL staining.Myeloperoxidase(MPO)activity was measured with MPO activity detection kit.IL-1βand TNF-αlevels were detected by ELISA.Macrophages and TNF-αin colon tissues were observed using immunofluorescence staining and confocal microscopy.Flow cytometry was performed to detect the changes in TLR4 expression on macrophages after stimulating mice with LA1 for 0,3,6 and 12 h.Moreover,TLR4 expression was also measured by Western blot after treating bone marrow-derived macrophages(BMDMs)with LA1 for 0,3,6 and 12 h.Unpaired t-test was used for statistical analysis.Results Compared with the DSS group,the LA1+DSS group presented lower mortality rate,greater body weight and longer colon and the differences between the two groups were statistically significant.Moreover,the LA1+DSS group showed lighter pathological damages,decreased percentage of apoptotic cells and suppressed MPO activity as compared with those of the DSS group.The number of macrophages and the relative concentrations of IL-1βand TNF-αin colon tissues were lower in the LA1+DSS group than in the DSS group,and the differences between the two groups were statistically significant.There was no significant difference in the total expression of TLR4 on macrophages before and after LA1 treatment.However,the mean flourscence indensity(MFI)of TLR4 was weaker on the LA1-treated macrophages than on the untreated macrophages.Conclusions LA1 could alleviate the DSS-induced experimental colitis in mice through suppressing the activation of TLR4 pathway on macrophages.This study provided a new insight and theoretical reference for understanding the pathogenesis of inflammatory bowel diseases.