甘露糖结合凝集素对肠上皮细胞凋亡的调控作用及机制研究

Research on the function and mechanism of mannose-binding lectin regulating intestinal epithelial apoptosis

目的探讨重组甘露糖结合凝集素(recombinant human mannose-binding lectin,rh MBL)对Caco-2细胞凋亡的影响及其机制。方法取14只C57小鼠,随机分为2组。经腹腔注射LPS,构建肠黏膜炎症损伤模型。通过免疫组化和RT-PCR观察甘露糖结合凝集素(MBL)的变化情况,使用TUNEL法检测肠上皮细胞凋亡情况。体外以0、5、10、20μg/m L的rh MBL处理Caco-2细胞,流式细胞术分析Caco-2细胞的凋亡情况,Western blotting和Real-Time PCR检测Caco-2细胞中Bax、Bcl-2基因和蛋白的表达情况,用Western blotting观察MAPK信号通路的变化并探讨其机制。结果经LPS刺激后,免疫组化和PCR观察到MBL在肠上皮细胞的表达增多,TUNEL染色显示肠上皮细胞凋亡增加。在体外不同浓度的rh MBL处理Caco-2细胞48 h后,20μg/m L rh MBL组的早期凋亡率高于其余组。选取20μg/m L的rh MBL以不同时间处理Caco-2细胞后,早期凋亡率增加。经20μg/m L处理48 h后,与对照组相比Bax/Bcl-2蛋白和基因表达比值升高,同时磷酸化P38、ERK蛋白表达增加。给予P38、ERK通路抑制剂后,与20μg/m L相比P38抑制剂能部分使Bax/Bcl-2蛋白表达比值降低,早期凋亡减少。结论高浓度rh MBL可促进肠上皮细胞细胞凋亡,并提示rh MBL可能是通过P38 MAPK途径参与细胞凋亡的调控。

Objective To observe the effect of recombinant human mannose-binding lectin （ rhMBL） on apoptosis of intestinal epithelial cells. Methods Fourteen C57 mice were randomly divided into two groups. The LPS mice received intra-peritoneal injection of LPS for indu-cing acute intestinal barrier injury. The expression of MBL protein and mRNA were observed by immuno-histochemisty and RT-PCR respec-tively. The apoptosis of intestinal epithelial cells was detected by tunel staining. In vitro, the Caco-2 cells were treated with 0、 5、10、20 μg/mLrhMBL, flow cytometry was used to detect the apoptosis rate of Caco-2 cells. The Bax and Bcl-2 protein and mRNA were detected by Western blotting and Real-Time PCR methods between various groups. MAPK signal proteins were detected by Western blotting method. Results The MBL and TUNEL staining were apparently increased under LPS condition. The apoptotic rates of Caco-2 cells which were trea-ted by different rhMBL concentrations after 48 h were increased in 20μg/mL rhMBL when compared with other groups. The apoptotic rates of Caco-2 cells which were treated by 20 μg/mL rhMBL after different time were increased in time-dependent manner. The results of Western blotting and Real-Time PCR assay showed that levels of Bax/Bcl-2 was increased in Caco-2 cells which were treated by 20μg/mL rhMBL af-ter 48h. Meanwhile, the phosphorylation of P38 and ERK were obviously increased in Caco-2 cells. The inhibitory effect of ERK and P38 in-hibitors were studied by Western blotting. Our current study showed that a block in P38 MAPK dependent cell death might contribute to the decrease in 20μg/mL MBL-mediated Caco-2 apoptosis. Conclusion High concentration of rhMBL may play an important role in the intesti-nal epithelial cell apoptosis, and P38 pathway may be involved in this regulation.