ATP介导的嘌呤能信号在阿尔茨海默病相关结肠运动障碍中的作用

目的:探讨ATP介导的嘌呤能信号在阿尔茨海默病(Alzheimer’s disease,AD)相关结肠运动障碍中的作用及其相关的分子机制。方法:(1)临床试验:收集我院20例AD患者进行研究,放射免疫法测定血浆中胃动素(motilin,MTL)、胆囊收缩素(cholecystokinin,CCK)、血管活性肠肽(vasoactive intestinal peptide,VIP)和一氧化氮(nitric oxide,NO)水平;高效液相色谱法(high-performance liquid chromatography,HPLC)检测血浆三磷酸腺苷(adenosine triphosphate,ATP)水平,同时对患者进行神经心理学检查并积分。(2)动物实验:利用AD小鼠进行Morris水迷宫实验,评估空间学习记忆功能;放射免疫法测定血浆中MTL、CCK、VIP和NO水平,HPLC法检测血浆ATP水平;免疫组织化学法检测乙酰胆碱转移酶(choline acetyltransferase,ChAT)、VIP、一氧化氮合酶(nitric oxide synthase,NOS)和ATP合酶的变化;Western blot和免疫组化法检测P2Y受体表达水平的变化。(3)离体实验:离体器官浴槽系统观察P2Y受体激动剂α,β-亚甲基ATP(α,β-methylene ATP,α,β-MeATP)对自发性及电刺激诱导的结肠平滑肌收缩的影响,细胞内微电极技术观察α,β-MeATP对结肠平滑肌细胞膜电位的影响。结果:与对照组比较,AD患者血浆的MTL和CCK水平明显降低(P0.01),NO和ATP水平显著升高(P0.05或P0.01),VIP无明显变化。患者简明精神状态检查积分(MMSE)降低(P0.05),AD评定量表-认知分量表(ADAS-Cog)积分、神经精神问卷(NPI)积分和AD协作研究日常能力量表(ADCS-ADL)积分均明显高于对照组(P0.01)。AD小鼠4~6 d逃逸潜伏期明显延长(P0.05),空间探索能力明显降低(P0.05),AD小鼠血浆中MTL和CCK水平明显降低(P0.01),NO和ATP水平显著升高(P0.05或P0.01),VIP无明显变化,AD小鼠结肠表达ATP合酶的水平明显上调(P0.05),但ChAT、VIP和NOS无明显改变,同时P2Y受体表达水平升高(P0.01)。体外实验表明,α,β-MeATP呈浓度依赖性抑制对照组和AD组小鼠结肠平滑肌自发性收缩(P0.05或P0.01),且这种抑制作用可被Na+通道阻断剂河豚毒素(tetrodotoxin,TTX)逆转(P0.05或P0.01),α,β-MeATP在100μmol/L时对AD小鼠自发性收缩的抑制作用更明显(P0.05),AD小鼠与正常小鼠比较,TTX对100μmol/L的α,β-MeATP的拮抗作用差异也有统计学显著性(P0.05)。在10 Hz电刺激诱导的结肠平滑肌收缩中,α,β-MeATP抑制正常小鼠和AD组小鼠的收缩(P0.05或P0.01),且40μmol/L和100μmol/L时对AD小鼠的抑制作用比正常小鼠明显(P0.05或P0.01)。平滑肌细胞膜电位实验显示,α,β-MeATP不影响结肠平滑肌膜电位(P0.05)。结论:AD患者和AD小鼠血浆中促进胃肠运动的激素MTL和CCK水平降低,抑制胃肠运动的激素NO水平升高,胃肠总体运动功能被抑制;AD小鼠血浆ATP水平升高,同时ATP嘌呤能神经元增加,P2Y受体表达上调;在AD发病中,ATP介导的嘌呤能信号可能通过抑制结肠平滑肌收缩,从而导致结肠运动功能障碍。

Role of purinergic signaling mediated by ATP in Alzheimer's disease-associated colonic motility disorder

AIM: To explore the role of purinergic signaling mediated by ATP in the Alzheimer's disease (AD)-related colon motility disorder and its related molecular mechanisms. METHODS: (1)Clinical trials:AD patients in our hospital were collected and studied. Radioimmunoassay was used for the determination of plasma motilin (MTL), cholecystokinin (CCK), vasoactive intestinal peptide (VIP) and nitric oxide (NO), and high-performance liquid chromatography (HPLC) was applied to test the level of adenosine triphosphate (ATP). The patients were assessed by neuropsychology and scored accordingly. (2)In animal experiments, AD mice received Morris water maze test, and the spatial learning and memory function were evaluated. The plasma levels of MTL, CCK, VIP and NO were examined by radioimmunoassay, and the level of ATP was measured by HPLC. Choline acetyltransferase (ChAT), VIP, nitric oxide synthase (NOS) and ATP synthase were detected by immunohistochemistry. Western blot and immunohistochemistry were used to detect the expression of P2Y receptor. (3)In vitro, organ bath was applied to observe the effect of α,β-methylene ATP (α,β-MeATP), an agonist of P2Y receptor, on both spontaneous and electrically evoked contraction of colonic smooth muscle strip, and the technique of intracellular microelectrode was applied to observe the effect of α,β-MeATP on the membrane potential of colonic smooth muscle cells. RESULTS: Compared with control group, the levels of MTL and CCK were decreased (P<0.01), and the levels of NO and ATP were increased (P < 0.05 or P < 0.01), while the VIP level was not changed. Mini-Mental State Examination (MMSE) score was decreased (P<0.05), Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) score, Neuropsychiatric Inventory (NPI) score and Alzheimer's Disease Cooperative Study-Activities of Daily Living Scale (ADCS-ADL) were all increased as compared with control group (P<0.01). The 4~6 d escape latency of APP/PS1 AD mice was significantly prolonged (P<0.05), and the space exploration ability distinctly reduced (P<0.05). In AD mice, the levels of MTL and CCK were decreased (P<0.01), and the levels of NO and ATP were increased (P<0.05 or P<0.01), while the VIP level was not changed. The protein expression of colonic ATP synthase was significantly increased (P<0.05), but the expression of ChAT, VIP and NOS was not changed. The expression of P2Y receptor was increased (P<0.01). The results of in vitro experiment displayed that α,β-MeATP, from 20 μmol/L to 100 μmol/L, inhibited the spontaneous contraction of colonic smooth muscle strip in the normal mice and AD mice (P < 0.05 or P < 0.01), and this inhibition was reversed by Na⁺ channel inhibitor tetrodotoxin (TTX) (P < 0.05 or P < 0.01). In addition, the effect of α,β-MeATP at 100 μmol/L on the AD mice was more obvious than that on the normal mice (P<0.05), and this inhibition was also antagonized by TTX (P < 0.05 or P < 0.01), pro-minent in AD group as compared with control group (P<0.05). In 10 Hz electrically evoked contraction of colonic smooth muscle strip, α,β-MeATP inhibited both the normal and AD mice (P < 0.05 or P < 0.01), while the inhibition was more obvious in the AD mice at the concentration of 40 μmol/L or 100 μmol/L (P < 0.05 or P < 0.01). CONCLUSION: AD patients and AD mice are accompanied by decreased MTL and CCK levels, and enhanced NO level, thus inducing colonic motor dysfunction along with AD. Meanwhile, ATP in plasma, purinergic neurons, and P2Y receptor expression are increased in the AD mice. Purinergic signaling mediated by ATP inhibits colonic smooth muscle strip contraction and further paralyzes the colonic movement function in AD.