探讨电针足三里穴对肠缺血再灌注损伤大鼠小肠黏膜上皮紧密连接蛋白闭锁小带蛋白(ZO)-1的调节作用。

按随机数字表法将30只SD大鼠分为模型组、电针足三里穴组、电针非经非穴组，每组10只。每组大鼠均于麻醉后沿腹正中线切口，分离肠系膜上动脉，用无损伤血管夹夹闭其起始部位，30 min后松夹恢复血流60 min，造成小肠缺血/再灌注损伤。电针足三里穴组于大鼠缺血后即刻行电针双侧足三里穴30 min，强度为2～3 mA，频率2～100 Hz；电针非经非穴组采用相同频率和强度于缺血后即刻刺激大鼠非经非穴(足三里穴外侧旁开0.5 cm)30 min；模型组不做任何治疗。于缺血再灌注损伤60 min时，以腹主动脉放血法处死各组大鼠，留取每只大鼠的血液和10 cm远端回肠组织。将留取的大鼠血液经10 000×g，4 ℃离心10 min后取上清得到血浆20 μL，全自动生化分析仪检测大鼠血浆脏器功能指标，包括谷丙转氨酶(GPT)、肌酸激酶同工酶(CK-MB)、尿素氮、肌酐；免疫荧光染色及蛋白质印迹法检测各组大鼠小肠黏膜上皮紧密连接蛋白ZO-1分布情况与表达水平。数据比较采用单因素方差分析和LSD-t检验。

缺血再灌注损伤60 min时，模型组大鼠GPT、CK-MB、尿素氮和肌酐水平分别为(88.1±11.6) μ/L、(482.3±69.7) μ/L、(11.6±3.7) mmol/L、(52.3±13.2) μmol/L，电针足三里穴组大鼠分别为(37.3±6.4) μ/L、(213.7±44.3) μ/L、(5.2±1.4) mmol/L、(30.1±5.7) μmol/L，电针非经非穴组大鼠分别为(85.7±13.4) μ/L、(460.2±72.3) μ/L、(10.8±4.2) mmol/L、(53.4±12.1) μmol/L，3组大鼠各指标比较，差异均有统计学意义(F＝69.4063、55.3611、10.9582、14.6817，P<0.05)。与模型组比较，电针足三里穴组大鼠血浆中GPT、CK-MB、尿素氮和肌酐水平与均降低，2组比较差异均有统计学意义(t＝12.126、10.285、5.116、4.883, P<0.05)，而电针非经非穴组大鼠血浆中GPT、CK-MB、尿素氮和肌酐水平与模型组相近，差异均无统计学意义(P>0.05)；电针足三里穴组大鼠血浆中GPT、CK-MB、尿素氮和肌酐水平均低于电针非经非穴组，比较差异均有统计学意义(t＝－10.307、－9.193、－4.000、－5.509, P<0.05)。免疫荧光染色结果显示，缺血再灌注损伤60 min时，模型组大鼠小肠黏膜上皮紧密连接蛋白ZO-1分布异常，连续性破坏，表达减少；电针足三里穴组大鼠小肠黏膜上皮紧密连接蛋白ZO-1分布密度较大、完整、连续；电针非经非穴组大鼠小肠黏膜上皮紧密连接蛋白ZO-1断裂，部分缺失，肠黏膜上皮紧密连接蛋白ZO-1荧光较弱、连续性消失。蛋白质印迹法检测结果可得，缺血再灌注损伤60 min时，电针足三里穴组大鼠小肠黏膜上皮紧密连接蛋白ZO-1蛋白表达(1.67±0.43)显著高于模型组(0.86±0.31)和电针非经非穴组(0.89±0.29)，比较差异均有统计学意义(t＝4.517、3.965，P<0.05)；电针非经非穴组大鼠小肠黏膜上皮紧密连接蛋白ZO-1蛋白表达与模型组比较，差异无统计学意义(P>0.05)。

电针足三里穴可有效调节肠黏膜上皮紧密连接蛋白ZO-1的正常分布与表达，能有效保护肠缺血再灌注损伤后肠黏膜屏障功能，减轻肠缺血再灌注后脏器功能损害。

To investigate the regulative effects of electroacupuncture at Zusanli point on the tight junction protein zonula occludens (ZO)-1 of small intestinal mucosal epithelium in rats with intestinal ischemia-reperfusion injury.

Thirty SD rats were divided into model group, electroacupuncture Zusanli group, electroacupuncture non-meridian non-acupoint group according to the random number table method, 10 rats in each group. After anesthesia, the rats in each group were cut along the median abdominal line, the superior mesenteric artery was separated, and its initial position was clamped with a non-invasive vascular clamp. After 30 minutes, the clamp was released to restore blood flow for 60 minutes, causing intestinal ischemia/reperfusion injury. In the electroacupuncture Zusanli group, electroacupuncture was performed at Zusanli point for 30 minutes immediately after ischemia, with an intensity of 2- 3 mA and a frequency of 2 - 100 Hz; in the electroacupuncture non-meridian non-acupoint group, rats were stimulated at non-meridian and non-acupoints with the same frequency and intensity immediately after ischemia (0.5 cm beside the lateral side of Zusanli point) for 30 minutes; the model group received no treatment. At 60 minutes of ischemia-reperfusion injury, the rats in each group were killed by abdominal aortic bloodletting, and the blood and 10 cm distal ileal tissue of each rat were retained. The collected rat blood was centrifuged at 10 000×g for 10 min at 4 ℃ and the supernatant was taken to obtain 20 μL of plasma. An automatic biochemical analyzer was used to detect the functional indexes of the rat plasma organs, including glutamic-pyruvic transaminase (GPT), creatine kinase isoenzyme (CK-MB), urea nitrogen and creatinine. Immunofluorescence staining and Western blotting were used to detect the distribution and expression of tight junction protein ZO-1 in the small intestinal mucosa of rats in each group. Data were compared by one-way ANOVA and LSD-t test.

At 60 minutes of ischemia-reperfusion injury, the levels of GPT, CK-MB, urea nitrogen and creatinine in the model group were (88.1±11.6) μ/L, (482.3±69.7) μ/L, (11.6±3.7) mmol/L, (52.3±13.2) μmol/L, electroacupuncture Zusanli group were (37.3±6.4) μ/L, (213.7±44.3) μ/L, (5.2±1.4) mmol/L, (30.1±5.7) μmol/L, electroacupuncture non-meridian non-acupoint group were (85.7±13.4) μ/L, (460.2±72.3) μ/L, (10.8±4.2) mmol/L, (53.4±12.1) μmol/L, respectively. There were statistically significant differences in the indexes of the three groups (F=69.4063, 55.3611, 10.9582, 14.6817; P < 0.05). Compared with the model group, the levels of GPT, CK-MB, urea nitrogen and creatinine in the plasma of rats in the electroacupuncture Zusanli group were all decreased, and the differences were statistically significant (t=12.126, 10.285, 5.116, 4.883; P< 0.05), while the levels of GPT, CK-MB, urea nitrogen and creatinine in the plasma of rats in the electroacupuncture non-meridian non-acupoint group were similar to the model group, and the differences between the 2 groups were not statistically significant (P> 0.05). The plasma GPT, CK-MB, urea nitrogen and creatinine levels of rats in the electroacupuncture Zusanli group were lower than those in the electroacupuncture non-meridian non-acupoint group, and the differences were statistically significant (t=-10.307, -9.193, -4.000, -5.509; P< 0.05). Immunofluorescence staining showed that at 60 minutes of ischemia-reperfusion injury, the distribution of tight junction protein ZO-1 in the small intestine mucosal epithelium of the rats in the model group was abnormal, the continuity was destroyed, and the expression was reduced. The distribution density of tight junction protein ZO-1 in small intestinal mucosal epithelium of rats in electroacupuncture Zusanli group was relatively dense, complete and continuous. In the electroacupuncture non-meridian non-acupoint group, the expression of ZO-1 protein in the small intestinal mucosal epithelium of rats was broken and partially missing, and the fluorescence of intestinal epithelial tight junction protein ZO-1 was weak and the continuity was disappeared. The results of Western blotting showed that at 60 minutes of ischemia-reperfusion injury, the expression of ZO-1 protein in the small intestinal mucosal epithelium of the rats of electroacupuncture Zusanli group (1.67±0.43) was significantly higher than that of model group (0.86±0.31) and electroacupuncture non-meridian non-acupoint group(0.89±0.29), and the differences were statistically significance (t=4.517, 3.965; P< 0.05). The expression of ZO-1 protein in intestinal tissue of electroacupuncture non-meridian non-acupoint group compared with the model group, the difference was no statistically significant (P>0.05).

Electroacupuncture at Zusanli point can effectively regulate the normal distribution and expression of tight junction protein ZO-1 in the intestinal mucosa, effectively protect the intestinal mucosal barrier function after intestinal ischemia-reperfusion injury, and reduce the damage of organ function after intestinal ischemia-reperfusion.