盐酸戊乙奎醚对大鼠小肠组织自噬的影响

Protective effects of penehycllidine hydrochloride on autophagy in small intestine caused by limb ischemia- reperfusion injury in rats

目的观察盐酸戊乙奎醚(长托宁)对大鼠肢体缺血再灌注损伤(LIRI)诱发小肠组织自噬的影响。方法采用大鼠肢体缺血再灌注损伤模型,选用雄性SD大鼠48只,按随机数字表法随机分为三组:假手术组(S组),缺血再灌注组(IR组),长托宁处理组(PHC组)。S组不阻断血流,IR、PHC组缺血3 h,各组于再灌注4 h后测定血清中乳酸脱氢酶(LDH)和肌酸激酶(CK)活性;荧光显微镜下观察小肠细胞自噬小体;聚合酶链式反应(PCR)检测自噬相关基因Beclin1、Atg5的m RNA表达;蛋白免疫印迹法(Western blot)检测自噬标志蛋白LC3蛋白的表达水平。结果 IR组血清LDH、CK分别为(5 231±463)、(3 673±231),与S组比较分别增加94%、161%(P<0.01);PHC组血清LDH、CK分别为(2 813±587)、(1 583±131),与S组比较分别增加5%、13%(P>0.05);与S组比较,IR组荧光显微镜下单位面积自噬小体明显增加,PHC组变化不明显;IR组Beclin1表达增加,与S组比较增加约127%(P<0.01),PHC组与S组比较增加约16%(P>0.05)。IR组Atg5表达增加,与S组比较增加约67%(P<0.05),PHC组与S组比较增加约14%(P>0.05)。IR组LC3表达增加,与S组比较LC3II与LC3I的比值分别增加约196%(P<0.01),PHC组与S组比较增加约18%(P>0.05)。结论盐酸戊乙奎醚可抑制肢体缺血再灌注损伤诱发大鼠小肠组织自噬水平的增加。

【Objective】To investigate the protective effects of penehyclidine hydrochloride on autophagy in small intestine induced by limb ischemia-reperfusion in rats. 【Methods】The model of limb ischemia-reperfusion injury was used to perform this experiment. Forty eight male SD rats were randomly divided into three groups： sham-operation group（group S）, limb ischemia-reperfusion group（group IR） and penehyclidine hydrochloride group（group PHC） and each group contained sixteen rats. In the group IR and the group PHC, bilateral limb ischemia was induced by rubber band application for 3 h, while in the group S bloodstream was not blocked. Lactate dehydrogenase（LDH） and creatine kinase（CK） activities in serum were measured in the samples taken from the inferior vena cava. The animals were killed and the small intestines were removed. The m RNA levels of beclin1 and Atg5 were measured by PCR,while LC3 protein level by Western blot.【Results】Compared with the group S, serum LDH and CK activities in the group IR increased significantly from（2,653 ± 512） to（5,231 ± 463） U/l（P 〈 0.01） and from（1,387 ± 109） to（3,673 ±231） U/l（P 〈 0.01）, respectively. Serum LDH and CK activities in the group PHC increased from（2,653 ± 512） to（2,813 ± 587） U/l（P 〉 0.05） and from（1,387 ± 109） U/l to（1,583 ± 131） U/l（P 〉 0.05）, respectively. Compared with the group S, autophagosomes in small intestines of the group IR increased significantly in a unit area under fluorescent microscope, while that in the group PHC didn’t. Compared with the group S, the level of beclin1 in small intestine increased about 127% in the group IR（P 〈0.01） and about 16% in the group PHC（P 〉 0.05）. Compared with the group S, the level of Atg5 in small intestine tissue increased about 67% in the group IR（P 〈 0.05） and about 14% in the group PHC（P 〉 0.05）. Compared with the group S, LC3II/LC3 I increased about 196% in the group IR（P 〈0.01） and about 18% in the group PHC（P 〉 0.05）. 【Conclusions】Penehyclidine hydrochloride can significantly attenuate the level of autophagy in small intestine, and protect small intestine from limb ischemia-reperfusion injury.