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Abstract：
BACKGROUND: The literature indicates that the enteric nervous system is affected in inflammatory bowel diseases (IBDs) and that the P2X7 receptor triggers neuronal death. However, the mechanism by which enteric neurons are lost in IBDs is unknown.
OBJECTIVE: To study the role of the caspase-3 and nuclear factor kappa B (NF-κB) pathways in myenteric neurons in a P2X7 receptor knockout (KO) mouse model of IBDs.
METHODS: Forty male wild-type (WT) C57BL/6 and P2X7 receptor KO mice were euthanized 24 h or 4 d after colitis induction by 2,4,6-trinitrobenzene sulfonic acid (colitis group). Mice in the sham groups were injected with vehicle. The mice were divided into eight groups (n = 5): The WT sham 24 h and 4 d groups, the WT colitis 24 h and 4 d groups, the KO sham 24 h and 4 d groups, and the KO colitis 24 h and 4 d groups. The disease activity index (DAI) was analyzed, the distal colon was collected for immunohistochemistry analyses, and immunofluorescence was performed to identify neurons immunoreactive (ir) for calretinin, P2X7 receptor, cleaved caspase-3, total caspase-3, phospho-NF-κB, and total NF-κB. We analyzed the number of calretinin-ir and P2X7 receptor-ir neurons per ganglion, the neuronal profile area (µm²), and corrected total cell fluorescence (CTCF).
RESULTS: Cells double labeled for calretinin and P2X7 receptor, cleaved caspase-3, total caspase-3, phospho-NF-κB, or total NF-κB were observed in the WT colitis 24 h and 4 d groups. The number of calretinin-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (2.10 ± 0.13 vs 3.33 ± 0.17, P < 0.001; 2.92 ± 0.12 vs 3.70 ± 0.11, P < 0.05), but was not significantly different between the KO groups. The calretinin-ir neuronal profile area was increased in the WT colitis 24 h group compared to the WT sham 24 h group (312.60 ± 7.85 vs 278.41 ± 6.65, P < 0.05), and the nuclear profile area was decreased in the WT colitis 4 d group compared to the WT sham 4 d group (104.63 ± 2.49 vs 117.41 ± 1.14, P < 0.01). The number of P2X7 receptor-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (19.49 ± 0.35 vs 22.21 ± 0.18, P < 0.001; 20.35 ± 0.14 vs 22.75 ± 0.51, P < 0.001), and no P2X7 receptor-ir neurons were observed in the KO groups. Myenteric neurons showed ultrastructural changes in the WT colitis 24 h and 4 d groups and in the KO colitis 24 h group. The cleaved caspase-3 CTCF was increased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (485949 ± 14140 vs 371371 ± 16426, P < 0.001; 480381 ± 11336 vs 378365 ± 4053, P < 0.001), but was not significantly different between the KO groups. The total caspase-3 CTCF, phospho-NF-κB CTCF, and total NF-κB CTCF were not significantly different among the groups. The DAI was recovered in the KO groups. Furthermore, we demonstrated that the absence of the P2X7 receptor attenuated inflammatory infiltration, tissue damage, collagen deposition, and the decrease in the number of goblet cells in the distal colon.
CONCLUSIONS: Ulcerative colitis affects myenteric neurons in WT mice but has a weaker effect in P2X7 receptor KO mice, and neuronal death may be associated with P2X7 receptor-mediated caspase-3 activation. The P2X7 receptor can be a therapeutic target for IBDs.
OBJECTIVE: To study the role of the caspase-3 and nuclear factor kappa B (NF-κB) pathways in myenteric neurons in a P2X7 receptor knockout (KO) mouse model of IBDs.
METHODS: Forty male wild-type (WT) C57BL/6 and P2X7 receptor KO mice were euthanized 24 h or 4 d after colitis induction by 2,4,6-trinitrobenzene sulfonic acid (colitis group). Mice in the sham groups were injected with vehicle. The mice were divided into eight groups (n = 5): The WT sham 24 h and 4 d groups, the WT colitis 24 h and 4 d groups, the KO sham 24 h and 4 d groups, and the KO colitis 24 h and 4 d groups. The disease activity index (DAI) was analyzed, the distal colon was collected for immunohistochemistry analyses, and immunofluorescence was performed to identify neurons immunoreactive (ir) for calretinin, P2X7 receptor, cleaved caspase-3, total caspase-3, phospho-NF-κB, and total NF-κB. We analyzed the number of calretinin-ir and P2X7 receptor-ir neurons per ganglion, the neuronal profile area (µm²), and corrected total cell fluorescence (CTCF).
RESULTS: Cells double labeled for calretinin and P2X7 receptor, cleaved caspase-3, total caspase-3, phospho-NF-κB, or total NF-κB were observed in the WT colitis 24 h and 4 d groups. The number of calretinin-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (2.10 ± 0.13 vs 3.33 ± 0.17, P < 0.001; 2.92 ± 0.12 vs 3.70 ± 0.11, P < 0.05), but was not significantly different between the KO groups. The calretinin-ir neuronal profile area was increased in the WT colitis 24 h group compared to the WT sham 24 h group (312.60 ± 7.85 vs 278.41 ± 6.65, P < 0.05), and the nuclear profile area was decreased in the WT colitis 4 d group compared to the WT sham 4 d group (104.63 ± 2.49 vs 117.41 ± 1.14, P < 0.01). The number of P2X7 receptor-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (19.49 ± 0.35 vs 22.21 ± 0.18, P < 0.001; 20.35 ± 0.14 vs 22.75 ± 0.51, P < 0.001), and no P2X7 receptor-ir neurons were observed in the KO groups. Myenteric neurons showed ultrastructural changes in the WT colitis 24 h and 4 d groups and in the KO colitis 24 h group. The cleaved caspase-3 CTCF was increased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (485949 ± 14140 vs 371371 ± 16426, P < 0.001; 480381 ± 11336 vs 378365 ± 4053, P < 0.001), but was not significantly different between the KO groups. The total caspase-3 CTCF, phospho-NF-κB CTCF, and total NF-κB CTCF were not significantly different among the groups. The DAI was recovered in the KO groups. Furthermore, we demonstrated that the absence of the P2X7 receptor attenuated inflammatory infiltration, tissue damage, collagen deposition, and the decrease in the number of goblet cells in the distal colon.
CONCLUSIONS: Ulcerative colitis affects myenteric neurons in WT mice but has a weaker effect in P2X7 receptor KO mice, and neuronal death may be associated with P2X7 receptor-mediated caspase-3 activation. The P2X7 receptor can be a therapeutic target for IBDs.
摘要：
背景：文献表明肠神经系统在炎症性肠病（IBD）中受到影响，并且P2X7受体触发神经元死亡。然而,肠神经元在IBD中丢失的机制尚不清楚。
目的：在IBDsP2X7受体敲除(KO)小鼠模型中研究caspase-3和核因子κB(NF-κB)通路在肌间神经元中的作用。
方法：40只雄性野生型（WT）C57BL/6和P2X7受体KO小鼠在2,4,6-三硝基苯磺酸诱导结肠炎后24h或4d实施安乐死（结肠炎组）。假手术组中的小鼠注射媒介物。将小鼠分为8组（n=5）：WTsham24h和4d组，WT结肠炎24h和4d组，KOSham24h和4d组，KO结肠炎24h和4d组。分析了疾病活动指数（DAI），收集远端结肠进行免疫组织化学分析，和免疫荧光进行鉴定神经元免疫反应性（ir）的钙，P2X7受体,裂解的caspase-3，总caspase-3，磷酸-NF-κB，和总NF-κB。我们分析了每个神经节的Calretinin-ir和P2X7受体-ir神经元的数量，神经元剖面面积(µm²)，和校正的总细胞荧光(CTCF)。
结果：细胞双重标记钙视网膜素和P2X7受体，裂解的caspase-3，总caspase-3，磷酸-NF-κB，在WT结肠炎24h和4d组中观察到或总NF-κB。与WTsham24h和4d组相比，WT结肠炎24h和4d组每个神经节的钙视网膜蛋白-ir神经元数量减少，分别（2.10±0.13vs3.33±0.17，P<0.001；2.92±0.12vs3.70±0.11，P<0.05），但KO组之间无显著差异。与WTsham24h组相比，WT结肠炎24h组的calretinin-ir神经元谱面积增加（312.60±7.85vs278.41±6.65，P<0.05），与WTsham4d组相比，WT结肠炎4d组的细胞核轮廓面积减少（104.63±2.49vs117.41±1.14，P<0.01）。与WTsham24h和4d组相比，WT结肠炎24h和4d组每个神经节的P2X7受体-ir神经元数量减少，分别（19.49±0.35vs22.21±0.18，P<0.001；20.35±0.14vs22.75±0.51，P<0.001），在KO组中未观察到P2X7受体-ir神经元。WT结肠炎24h和4d组和KO结肠炎24h组的肌肠神经元显示超微结构变化。与WTsham24h和4d组相比，WT结肠炎24h和4d组的caspase-3CTCF增加，分别（485949±14140vs371371±16426，P<0.001；480381±11336vs378365±4053，P<0.001），但KO组之间无显著差异。总caspase-3CTCF，磷酸-NF-κBCTCF，和总NF-κBCTCF在各组之间没有显着差异。在KO组中回收DAI。此外，我们证明了P2X7受体的缺失减轻了炎症浸润，组织损伤,胶原蛋白沉积，远端结肠杯状细胞数量减少。
结论：溃疡性结肠炎影响WT小鼠的肌间神经元，但对P2X7受体KO小鼠的影响较弱，神经元死亡可能与P2X7受体介导的caspase-3激活有关。P2X7受体可以是IBD的治疗靶标。
目的：在IBDsP2X7受体敲除(KO)小鼠模型中研究caspase-3和核因子κB(NF-κB)通路在肌间神经元中的作用。
方法：40只雄性野生型（WT）C57BL/6和P2X7受体KO小鼠在2,4,6-三硝基苯磺酸诱导结肠炎后24h或4d实施安乐死（结肠炎组）。假手术组中的小鼠注射媒介物。将小鼠分为8组（n=5）：WTsham24h和4d组，WT结肠炎24h和4d组，KOSham24h和4d组，KO结肠炎24h和4d组。分析了疾病活动指数（DAI），收集远端结肠进行免疫组织化学分析，和免疫荧光进行鉴定神经元免疫反应性（ir）的钙，P2X7受体,裂解的caspase-3，总caspase-3，磷酸-NF-κB，和总NF-κB。我们分析了每个神经节的Calretinin-ir和P2X7受体-ir神经元的数量，神经元剖面面积(µm²)，和校正的总细胞荧光(CTCF)。
结果：细胞双重标记钙视网膜素和P2X7受体，裂解的caspase-3，总caspase-3，磷酸-NF-κB，在WT结肠炎24h和4d组中观察到或总NF-κB。与WTsham24h和4d组相比，WT结肠炎24h和4d组每个神经节的钙视网膜蛋白-ir神经元数量减少，分别（2.10±0.13vs3.33±0.17，P<0.001；2.92±0.12vs3.70±0.11，P<0.05），但KO组之间无显著差异。与WTsham24h组相比，WT结肠炎24h组的calretinin-ir神经元谱面积增加（312.60±7.85vs278.41±6.65，P<0.05），与WTsham4d组相比，WT结肠炎4d组的细胞核轮廓面积减少（104.63±2.49vs117.41±1.14，P<0.01）。与WTsham24h和4d组相比，WT结肠炎24h和4d组每个神经节的P2X7受体-ir神经元数量减少，分别（19.49±0.35vs22.21±0.18，P<0.001；20.35±0.14vs22.75±0.51，P<0.001），在KO组中未观察到P2X7受体-ir神经元。WT结肠炎24h和4d组和KO结肠炎24h组的肌肠神经元显示超微结构变化。与WTsham24h和4d组相比，WT结肠炎24h和4d组的caspase-3CTCF增加，分别（485949±14140vs371371±16426，P<0.001；480381±11336vs378365±4053，P<0.001），但KO组之间无显著差异。总caspase-3CTCF，磷酸-NF-κBCTCF，和总NF-κBCTCF在各组之间没有显着差异。在KO组中回收DAI。此外，我们证明了P2X7受体的缺失减轻了炎症浸润，组织损伤,胶原蛋白沉积，远端结肠杯状细胞数量减少。
结论：溃疡性结肠炎影响WT小鼠的肌间神经元，但对P2X7受体KO小鼠的影响较弱，神经元死亡可能与P2X7受体介导的caspase-3激活有关。P2X7受体可以是IBD的治疗靶标。
