Objective To explore the mechanism of the bone morphogenetic protein-4 (BMP-4) promote apoptosis of intestinal intraepithelial lymphocytes through downregulating IL-7/IL-7R signaling after I/R.Methods Mouse intestinal I/R model was established.The mice were randomly divided into I/R group and sham operation group.IL-7 protein expression in IECs (immunofluorescence) and IL-7 receptor(CD127) and phosphorylation of STAT5 proteins in IELs (flow cytometry) were detected.Western-bolt detect the change of IL-7protein expression after treating the IEC-6 with exogenous BMP-4 protein for 6 hour.IELs were stimulated with exogenous BMP4 and BMP4 antagonist NOGGIN protein.IL-7 receptor (CD127) and P-STAT5 proteins expression were measured by flow cytometry.IEL was stimulated with exogenous BMP-4 and IL-7 protein.The apoptosis was detected.Results The IL-7 protein secretion of I/R group significantly decreased,compared with that of sham group after I/R in IECs.The IL-7 receptor (CD127) and P-STAT5 expression of I/R group were also decreased after I/R in IELs(P＜0.05).Under condition of exogenous BMP-4 stimulation,the expression of IL-7 protein was decreased in IEC-6,and the expression of CD127 and P-STAT5 protein also decreased in IELs(P＜0.05).The apoptosis of IELs were significantly increased with BMP-4 protein stimulation than the control group(P＜0.05).However,IL-7 protein can promote the apoptosis of IELs(P＜0.05).Conclusion The IECs derived BMP-4 protein downregulates IL-7/IL-7R signaling pathway in I/R.The protective factors of IELs are decreased,and the apoptosis rate of IELs is increased.%目的 探讨小鼠小肠在肠缺血再灌注(I/R)条件下骨形成蛋白(BMP-4)通过下调IL-7/IL-7R信号通路促进IELs凋亡的机制.方法 建立小鼠小肠I/R损伤模型,随机分为手术组(I/R组)、假手术组(Sham组),每组各16只,免疫荧光检测IECs中IL-7蛋白和流式细胞术检测IELs中IL-7受体(CD127)及STAT5蛋白磷酸化水平变化;外源性BMP-4蛋白刺激IEC-6培养6小时后,Western blot检测IEC-6中IL-7蛋白表达变化;分离培养正常小鼠IELs,分别加入外源性BMP-4蛋白及BMP4拮抗剂NOGGIN蛋白刺激,流式细胞术观察BMP4对IELs中IL-7受体(CD127)及STAT5蛋白磷酸化变化;分离培养正常小鼠IELs,分别加入外源性BMP-4蛋白及IL-7蛋白刺激,观察IELs凋亡率的变化.结果 在I/R刺激下,IECs中IL-7蛋白表达I/R组较Sham组明显减少(P＜0.05);IELs中IL-7受体(CD127)及STAT5蛋白磷酸化水平均较Sham组表达减少(P＜0.05);体外培养实验发现:IECs中IL-7蛋白表达在BMP-4刺激下明显减少,IELs单独培养给予BMP4蛋白刺激后CD127及P-STAT5较Sham组减少(P＜0.05);给予BMP特异性拮抗剂RNOGGIN可逆转BMP对IL-7/IL-7R信号通路的抑制作用;BMP-4促进IELs的凋亡率增加(P＜0.05),给予外源性IL-7蛋白刺激后,有效的抑制IELs的凋亡率增加(P＜0.05),BMP-4抑制IL-7对IELs的增殖作用,促进IELs的凋亡.结论 小肠在I/R条件下,肠上皮细胞中BMP-4蛋白抑制IL-7/IL-7R信号通路,使IELs的保护性细胞因子减少,从而促进IELs的凋亡率增加,进一步加重肠免疫屏障损伤.
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