Abstract：
OBJECTIVE: To observe the effect of electroacupuncture (EA) on urodynamics and Raf/MEK/ERK signaling pathway in spine cord tissue of rats after suprasacral spinal cord injury (SSCI), so as to explore its possible mechanism in improving bladder function in rats with detrusor hyperreflexia after SSCI.
METHODS: Female SD rats were randomly divided into blank, sham operation, model, EA and EA+PD98059 groups, with 12 rats in each group. Thorax (T) 10 spinal cord transection was performed by surgery. Rats in the EA group were given EA (10 Hz/50 Hz, 20 min) at \"Ciliao\" (BL32), \"Zhongji\" (CV3), \"Sanyinjiao\" (SP6) and \"Dazhui\" (GV14) once daily for 7 d. Rats of the EA+PD98059 group received intraperitoneal injection of PD98059 (5 mg/kg) 2 h before EA intervention. The urodyna-mics was used to measure the base pressure, leak point pressure, maximum pressure, maximum capacity and comp-liance of bladder, and the morphology of bladder detrusor tissue was observed with HE staining. The TUNEL staining was used to detect the cell apoptosis of the spinal cord tissue. The expression levels of exchange protein directly activated by cAMP 2 (Epac2), Rap, phosphorylated rapidly accelerated fibrosarcoma (p-Raf), phosphorylated mitogen-activated extracellular signal-regulated kinase (p-MEK), phosphorylated extracellular signal regulated kinase 1 and 2 (p-ERK1/2), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) were determined by Western blot.
RESULTS: Compared with the sham operation group, the base pressure, leak point pressure and maximum pressure of bladder were significantly increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), the cell apoptosis rate of spinal cord tissue was increased (P<0.01), and the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were decreased (P<0.01), while the expression level of Bax protein was increased (P<0.01) in the model group. After the treatment and compared with the model group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate of spinal cord tissue, the expression level of Bax protein were decreased (P<0.05) in the EA group, while the maximum bladder capacity and bladder compliance, the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were all increased (P<0.05, P<0.01). In comparison with the EA group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate, the expression level of Bax protein were significantly increased (P<0.05), whereas the maximum bladder capacity, bladder compliance, and the expression levels of p-MEK, p-ERK1/2, and Bcl-2 protein were decreased (P<0.05) in the EA+PD98059 group. Results of HE staining showed disordered transitional epithelial cells and destroyed lamina propria in bladder detrusor tissue, with the infiltration of monocytes in the model group, which was obviously milder in both EA and EA+PD98059 groups, especially in the EA group.
CONCLUSIONS: EA can improve the bladder function in detrusor hyperreflexia rats after SSCI, which may be related to its effect in up-regulating Epac2 and Rap, activating the Raf-MEK-ERK pathway, and reducing the cell apoptosis of spinal cord tissue.
目的: 观察电针对骶上脊髓损伤（SSCI）后逼尿肌反射亢进型大鼠尿流动力学及脊髓组织中丝氨酸/苏氨酸蛋白激酶（Raf）与丝裂原活化的细胞外信号调节激酶（MEK）及细胞外信号调节激酶1/2（ERK1/2）信号通路的影响，探讨电针改善SSCI后逼尿肌反射亢进型大鼠膀胱功能的可能机制。方法: 72只SD大鼠随机分为空白组和假手术组各12只，其余48只大鼠采用脊髓横断法造模，在符合模型标准的大鼠中随机抽取36只分为模型组、电针组和电针+PD98059组各12只。电针组大鼠于“次髎”“中极”“三阴交”“大椎”给予电针治疗，20 min/次，1次/d，连续7 d；电针+PD98059组大鼠在同样电针干预前2 h给予腹腔注射PD98059（5 mg/kg）。采用尿流动力学检测大鼠膀胱基础压、漏尿点压、最大压力、最大容量及顺应性，HE染色观察大鼠膀胱逼尿肌组织形态，用TUNEL法测定脊髓组织细胞凋亡情况，用Western blot法检测脊髓组织中环磷酸腺苷直接激活交换蛋白2（Epac2）、小分子G蛋白Rap、磷酸化（p）-Raf、p-MEK、p-ERK 1/2、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关蛋白X（Bax）的表达。结果: 与假手术组比较，模型组大鼠膀胱基础压、漏尿点压、最大压力均升高（P<0.01），而膀胱最大容量减少（P<0.01），膀胱顺应性降低（P<0.01），脊髓组织细胞凋亡率升高（P<0.01），脊髓组织Epac2、Rap、p-Raf、p-MEK、p-ERK1/2及Bcl-2蛋白表达水平降低（P<0.01），Bax蛋白表达水平升高（P<0.01）。与模型组比较，电针组大鼠膀胱基础压、漏尿点压、最大压力均降低（P<0.05），膀胱最大容量增加（P<0.05），膀胱顺应性升高（P<0.05），脊髓组织细胞凋亡率降低（P<0.05），脊髓组织Epac2、Rap、p-Raf、p-MEK、p-ERK1/2及Bcl-2蛋白表达水平升高（P<0.05，P<0.01），Bax蛋白表达水平降低（P<0.05）。与电针组比较，电针＋PD98059组大鼠膀胱基础压、漏尿点压、最大压力均升高（P<0.05），膀胱最大容量减少（P<0.05），膀胱顺应性降低（P<0.05），脊髓组织细胞凋亡率升高（P<0.05），脊髓组织p-MEK、p-ERK1/2及Bcl-2蛋白表达水平均降低（P<0.05），Bax蛋白表达水平升高（P<0.05）。HE染色结果显示，空白组和假手术组大鼠膀胱逼尿肌组织细胞排列紧密整齐，细胞形态完整；模型组大鼠膀胱逼尿肌移行上皮细胞排列紊乱，固有膜被破坏，伴单核细胞浸润；与模型组比较，电针组膀胱逼尿肌组织弹性纤维增生程度减轻，肌纤维排列层次较清晰，可观察到更多形态饱满的完整细胞；与电针组比较，电针＋PD98059组逼尿肌组织肌纤维排列层次欠清晰，细胞间隙大。结论: 电针可改善SSCI后逼尿肌反射亢进型大鼠的膀胱功能，其机制可能与电针上调脊髓Epac2、Rap，启动Raf-MEK-ERK级联反应，减少细胞凋亡，改善膀胱神经支配有关。.
METHODS: Female SD rats were randomly divided into blank, sham operation, model, EA and EA+PD98059 groups, with 12 rats in each group. Thorax (T) 10 spinal cord transection was performed by surgery. Rats in the EA group were given EA (10 Hz/50 Hz, 20 min) at \"Ciliao\" (BL32), \"Zhongji\" (CV3), \"Sanyinjiao\" (SP6) and \"Dazhui\" (GV14) once daily for 7 d. Rats of the EA+PD98059 group received intraperitoneal injection of PD98059 (5 mg/kg) 2 h before EA intervention. The urodyna-mics was used to measure the base pressure, leak point pressure, maximum pressure, maximum capacity and comp-liance of bladder, and the morphology of bladder detrusor tissue was observed with HE staining. The TUNEL staining was used to detect the cell apoptosis of the spinal cord tissue. The expression levels of exchange protein directly activated by cAMP 2 (Epac2), Rap, phosphorylated rapidly accelerated fibrosarcoma (p-Raf), phosphorylated mitogen-activated extracellular signal-regulated kinase (p-MEK), phosphorylated extracellular signal regulated kinase 1 and 2 (p-ERK1/2), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) were determined by Western blot.
RESULTS: Compared with the sham operation group, the base pressure, leak point pressure and maximum pressure of bladder were significantly increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), the cell apoptosis rate of spinal cord tissue was increased (P<0.01), and the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were decreased (P<0.01), while the expression level of Bax protein was increased (P<0.01) in the model group. After the treatment and compared with the model group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate of spinal cord tissue, the expression level of Bax protein were decreased (P<0.05) in the EA group, while the maximum bladder capacity and bladder compliance, the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were all increased (P<0.05, P<0.01). In comparison with the EA group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate, the expression level of Bax protein were significantly increased (P<0.05), whereas the maximum bladder capacity, bladder compliance, and the expression levels of p-MEK, p-ERK1/2, and Bcl-2 protein were decreased (P<0.05) in the EA+PD98059 group. Results of HE staining showed disordered transitional epithelial cells and destroyed lamina propria in bladder detrusor tissue, with the infiltration of monocytes in the model group, which was obviously milder in both EA and EA+PD98059 groups, especially in the EA group.
CONCLUSIONS: EA can improve the bladder function in detrusor hyperreflexia rats after SSCI, which may be related to its effect in up-regulating Epac2 and Rap, activating the Raf-MEK-ERK pathway, and reducing the cell apoptosis of spinal cord tissue.
目的: 观察电针对骶上脊髓损伤（SSCI）后逼尿肌反射亢进型大鼠尿流动力学及脊髓组织中丝氨酸/苏氨酸蛋白激酶（Raf）与丝裂原活化的细胞外信号调节激酶（MEK）及细胞外信号调节激酶1/2（ERK1/2）信号通路的影响，探讨电针改善SSCI后逼尿肌反射亢进型大鼠膀胱功能的可能机制。方法: 72只SD大鼠随机分为空白组和假手术组各12只，其余48只大鼠采用脊髓横断法造模，在符合模型标准的大鼠中随机抽取36只分为模型组、电针组和电针+PD98059组各12只。电针组大鼠于“次髎”“中极”“三阴交”“大椎”给予电针治疗，20 min/次，1次/d，连续7 d；电针+PD98059组大鼠在同样电针干预前2 h给予腹腔注射PD98059（5 mg/kg）。采用尿流动力学检测大鼠膀胱基础压、漏尿点压、最大压力、最大容量及顺应性，HE染色观察大鼠膀胱逼尿肌组织形态，用TUNEL法测定脊髓组织细胞凋亡情况，用Western blot法检测脊髓组织中环磷酸腺苷直接激活交换蛋白2（Epac2）、小分子G蛋白Rap、磷酸化（p）-Raf、p-MEK、p-ERK 1/2、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关蛋白X（Bax）的表达。结果: 与假手术组比较，模型组大鼠膀胱基础压、漏尿点压、最大压力均升高（P<0.01），而膀胱最大容量减少（P<0.01），膀胱顺应性降低（P<0.01），脊髓组织细胞凋亡率升高（P<0.01），脊髓组织Epac2、Rap、p-Raf、p-MEK、p-ERK1/2及Bcl-2蛋白表达水平降低（P<0.01），Bax蛋白表达水平升高（P<0.01）。与模型组比较，电针组大鼠膀胱基础压、漏尿点压、最大压力均降低（P<0.05），膀胱最大容量增加（P<0.05），膀胱顺应性升高（P<0.05），脊髓组织细胞凋亡率降低（P<0.05），脊髓组织Epac2、Rap、p-Raf、p-MEK、p-ERK1/2及Bcl-2蛋白表达水平升高（P<0.05，P<0.01），Bax蛋白表达水平降低（P<0.05）。与电针组比较，电针＋PD98059组大鼠膀胱基础压、漏尿点压、最大压力均升高（P<0.05），膀胱最大容量减少（P<0.05），膀胱顺应性降低（P<0.05），脊髓组织细胞凋亡率升高（P<0.05），脊髓组织p-MEK、p-ERK1/2及Bcl-2蛋白表达水平均降低（P<0.05），Bax蛋白表达水平升高（P<0.05）。HE染色结果显示，空白组和假手术组大鼠膀胱逼尿肌组织细胞排列紧密整齐，细胞形态完整；模型组大鼠膀胱逼尿肌移行上皮细胞排列紊乱，固有膜被破坏，伴单核细胞浸润；与模型组比较，电针组膀胱逼尿肌组织弹性纤维增生程度减轻，肌纤维排列层次较清晰，可观察到更多形态饱满的完整细胞；与电针组比较，电针＋PD98059组逼尿肌组织肌纤维排列层次欠清晰，细胞间隙大。结论: 电针可改善SSCI后逼尿肌反射亢进型大鼠的膀胱功能，其机制可能与电针上调脊髓Epac2、Rap，启动Raf-MEK-ERK级联反应，减少细胞凋亡，改善膀胱神经支配有关。.
摘要：
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