汉黄芩素通过诱导中性粒细胞凋亡治疗小鼠结肠炎

Wogonin treats colitis mice by inducing apoptosis of neutrophils

目的观察汉黄芩素对葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎的影响并探讨作用机制。方法将18只C57BL/6小鼠随机分为对照组、模型组和治疗组,每组6只。对照组正常饮水,其他2组连续给予含2.5%DSS的饮用水7 d,治疗组在喂水第2天和第4天腹腔注射汉黄芩素。第8天处死小鼠并取材,通过测量小鼠结肠长度和HE染色评估小鼠结肠损伤和炎症水平,利用免疫荧光检测小鼠结肠组织中性粒细胞的浸润数量。另外,以25、50、100μmol/L汉黄芩素体外干预小鼠骨髓中性粒细胞,阴性对照组不予任何干预。采用流式细胞术检测中性粒细胞的凋亡,Western blot检测中性粒细胞抗凋亡蛋白髓细胞白血病-1(Mcl-1)和细胞外信号调节激酶(ERK)表达变化。结果与模型组比较,治疗组的小鼠结肠明显较长[(7.80±0.21)cm比(6.43±0.10)cm,P<0.01)],结肠组织病理损伤评分明显降低[(6.83±0.98)分比(14.33±1.03)分,P<0.01)],结肠中性粒细胞浸润显著减少[(8.52±0.15)个/低倍镜视野比(29.43±0.43)个/低倍镜视野,P<0.01)]。阴性对照组以及25、50、100μmol/L汉黄芩素组中性粒细胞的凋亡率分别为6.41%±0.51%、14.01%±0.81%、20.89%±0.82%、24.23%±0.29%;随着汉黄芩素浓度增高,中性粒细胞凋亡率明显升高,组间差异具有统计学意义(均P<0.01)。与阴性对照组比较,25、50、100μmol/L汉黄芩素组中性粒细胞磷酸化ERK表达均明显减少(均P<0.05)。随着汉黄芩素浓度升高,中性粒细胞Mcl-1蛋白表达逐渐减少(均P<0.05)。结论汉黄芩素可浓度依赖性诱导小鼠中性粒细胞凋亡,减少结肠组织中性粒细胞浸润,减轻肠道损伤,上述作用可能通过抑制ERK磷酸化和浓度依赖性下调Mcl-1表达来实现的。

Objective To observe the influence of wogonin on colitis mice induced by dextran sulfate sodium(DSS)and explore the related mechanism.Methods Eighteen C57BL/6 mice were randomly and equally divided into the control group,the model group and the treatment group.The water was given normally to mice in control group,and the 2.5%DSS drinking water was given to mice of other two groups for 7 days.Wogonin via intraperitoneal injection was administrated in the mice of treatment group on the second and the fourth day.The mice were sacrificed on the eighth day and specimens were collected.The pathological damage and inflammation degree of mice colon were evaluated by measuring the length of colon and using HE staining.Immunofluorescence was used to detect the infiltration of neutrophils in mice colon tissue.Wogonin of 25,50,100μmol/L was applied to handle neutrophils from mice marrow tissue in vitro,and there was no treatment in the negative control group.The flow cytometry was used to detect the apoptosis of neutrophil.Western blot was used to detect the expressions of anti-apoptotic protein myeloid cell leukelia-1(Mcl-1)and extracellular signal-regulated kinase(ERK).Results Compared with the model group,the mice colon length in the treatment group was significantly longer[(7.80±0.21)cm vs.(6.43±0.10)cm,P<0.01],the pathological damage score of the colon tissue was significantly lower[(6.83±0.98)points vs.(14.33±1.03)points,P<0.01],the number of infiltrative neutrophils in the colon of mice was significantly lower[(8.52±0.15)neutrophils per low power field vs.(29.43±0.43)neutrophils per low power field,P<0.01].The apoptosis rate of neutrophils were 6.41%±0.51%,14.01%±0.81%,20.89%±0.82%,24.23%±0.29%in negative control group and 25,50,100μmol/L wogonin groups.The apotosis rate of neutrophils increased constantly with the concentration of wogonin increasing gradually and there were significant differences among any two groups(P<0.01).Compared with the negative control group,the phosphorylated ERK expressions of neutrophils in 25,50,100μmol/L wogonin groups were decreased obviously(all P<0.05).The Mcl-1 expression of neutrophils declined constantly with the concentration of wogonin increasing gradually.Conclusion Wogonin can induce the apoptosis of neutrophils in concentration-dependent manner,reduce the infiltration of neutrophils and relieve the intestinal damage in colon tissue of colitis mice,which may be regulated by the inhibition of ERK phosphorylation and decreased expression of Mcl-1 in concentration-dependent manner.