腺苷A3受体活化对溃疡性结肠炎患者结肠黏膜5-羟色胺的影响

Effects of adenosine A3 receptor activation on colonic mucosal serotonin in patients with ulcerative colitis

目的探讨腺苷A3受体(A3AR)对溃疡性结肠炎(UC)患者结肠黏膜5-羟色胺(5-HT)的影响及其机制。方法选择2016年6月—2018年1月在香港大学深圳医院就诊并行结肠镜检查的患者,其中活动期UC患者18例(UC组),健康体格检查者11名(对照组)。经结肠镜检查获取UC组和对照组的结肠黏膜组织,取部分UC组的结肠黏膜组织,在其培养基中加入1-[2-氯-6-[[(3-碘苯基)甲基]氨基]-9H-嘌呤-9-基]-1-脱氧-N-甲基-β-D-呋喃尿酰胺(2-Cl-IB-MECA,100 nmol/L)处理24 h(UC+2-Cl-IB-MECA组),对照组和另一部分UC组的结肠黏膜组织仅以培养基孵育。采用ELISA法测定结肠黏膜TNF-α和IL-1β水平、5-HT含量和释放量,采用免疫荧光法检测A3AR、5-HT和5-HT合成酶色氨酸羟化酶1(TPH1)在结肠黏膜中的表达,荧光显微镜下观察肠嗜铬细胞(EC细胞)数,采用Western印迹法定量检测A3AR和TPH1蛋白质的表达。结果 UC组结肠黏膜A3AR蛋白质相对表达量为1.27±0.19,显著低于对照组的2.43±0.25(P<0.01)。UC组和UC+2-Cl-IB-MECA组的TNF-α和IL-1β水平均显著高于对照组(P值均<0.01),而UC+2-Cl-IB-MECA组的TNF-α和IL-1β水平均显著低于UC组(P值均<0.01)。UC组和UC+2-Cl-IB-MECA组的EC细胞数和5-HT含量均显著多于对照组(P值均<0.01),而UC+2-Cl-IB-MECA组EC细胞数和5-HT含量均显著少于UC组(P值均<0.01)。UC组和UC+2-Cl-IB-MECA组结肠黏膜TPH1蛋白质水平均显著高于对照组(P值均<0.01),而UC+2-Cl-IB-MECA组结肠黏膜TPH1蛋白质水平显著低于UC组(P值均<0.01)。UC组和UC+2-Cl-IB-MECA组结肠黏膜5-HT释放量均显著高于对照组(P值均<0.01),而UC+2-Cl-IB-MECA组结肠黏膜5-HT释放量显著低于UC组(P<0.01)。结论 A3AR的激活可减轻UC患者的结肠黏膜炎性反应,其抗炎作用可能通过减少结肠黏膜5-HT的合成和释放而介导。A3AR可作为UC的治疗靶点。

Objective To investigate the effect of adenosine A3 receptor(A3 AR)on serotonin(5-HT)in colonic mucosa of patients with ulcerative colitis(UC)and its mechanism.Methods From June 2016 to January 2018,18 patients with active UC(UC group)and 11 healthy subjects(control group)who underwent colonoscopy were recruited in this study.Colon mucosa of both groups were obtained by colonoscopy.Some colon mucosas of UC group were treated with 1-[2-Chloro-6[[(3-iodophenyl)methyl]amino]-9 H-purin-9-yl]-1-deoxy-N-methyl-β-D-ribofuranuroamide(2-Cl-IB-MECA,100 nmol/L)for 24 h(UC+2-Cl-IB-MECA group).Colon mucosa of control group and UC group was incubated in culture medium.The levels of tumor necrosis factor(TNF)-αand interleukin(IL)-1β,5-HT content and release from colonic mucosa were measured by enzyme-linked immunosorbent assay(ELISA).The expression of A3 AR,5-HT and 5-HT synthase tryptophan hydroxylase 1(TPH1)in colonic mucosa were detected by immunofluorescence.The number of enterochromaffin cells(EC)was observed under fluorescence microscope.The protein expression of A3 AR and TPH1 were quantitatively detected by immunoblotting.Results The expression of A3 AR protein in colonic mucosa of UC group was 1.27±0.19,which was significantly lower than that of control group(2.43±0.25,P<0.01).The levels of TNF-αand IL-1βin UC group and UC+2-Cl-IB-MECA group were significantly higher than those in control group(all P<0.01),while the levels of TNF-αand IL-1βin UC+2-Cl-IB-MECA group were significantly lower than those in UC group(both P<0.01).EC count and 5-HT content in UC group and UC+2-Cl-IB-MECA group were significantly more than those in control group(all P<0.01),while EC count and 5-HT content in UC+2-Cl-IB-MECA group were significantly less than those in UC group(both P<0.01).The TPH1 protein level and 5-HT release in UC group and UC+2-Cl-IB-MECA group were significantly higher than those in control group(all P<0.01),while the TPH1 protein level and 5-HT release in UC+2-Cl-IB-MECA group were significantly lower than those in UC group(both P<0.01).Conclusion The activation of A3 AR can alleviate colonic mucosal inflammation in UC patients,and its anti-inflammatory effect may be mediated by reduced synthesis and release of 5-HT in colonic mucosa.A3 AR may provide an alternative therapeutic target for UC.