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[电针及脑内注射血管内皮生长因子对脑缺血再灌注损伤大鼠半胱天冬酶12、半胱天冬酶3及相关基因的影响]

[Effects of Electroacupuncture and Intracerebral Injection of VEGF on Caspase12, Caspase3, and Genes in Rats with Cerebral Ischemia-Reperfusion Injury].

作者信息

Wu Jia-Peng, Li Xue-Zhi, Wang Ying, Ma Lin, Yao Tai-Wan, Zhang Yong-Yue, Long Fei

机构信息

College of Traditional Chinese Medicine, Chongqing Medical University, Chongqing 401331, China.

Chinese Medicine Hospital of Jiang Jin Chongqing, Chongqing 402260, China.

出版信息

Sichuan Da Xue Xue Bao Yi Xue Ban. 2019 Jan;50(1):34-39.

Abstract

OBJECTIVE

To determine the effects of electroacupuncture (EA) and intracerebral injection of vascular endothelial growth factor (VEGF) on caspase12, caspase3, and glucose regulated protein 78 kD (GRP78) genes of rats with cerebral ischemia reperfusion injury.

METHODS

60 SD rats were randomly divided into sham-operation group, model group, EA group and EA+VEGF group with 15 rats in each group. Middle cerebral artery occlusion (MCAO) method was used to establish the model of cerebral ischemia reperfusion injury. Electro-acupuncture intervention was introduced 1 day after the injury in the EA group and EA+VEGF group: 30 minutes each session and once a day for a total of 14 d [acupoint selection: Baihui (GV 20), Quchi(Li 11), Zusanli (ST36)]. The rats in the EA+VEGF group were also injected with 10 μL of VEGF165 (0.025 μg/μL) into the lateral ventricle after the first session of EA. Five rats in each group were sacrificed after obtaining a neurological function score (mNSS) at day 0 (1 d after modeling, before EA intervention), day 7 and day 14, respectively. Nissl staining was used to observe the histomorphology of cerebral infarction areas. Immunohistochemistry was used to CM(155mm]detect GRP78 activity in the ischemic brain tissues. Real-time fluorescence quantitative PCR (real-time PCR) was used to detect the expressions of caspase12, caspase3 and mRNA in the ischemic brain tissues.

RESULTS

Compared with the sham-operation group, rats in the model group had higher mNSS scores ( <0.05), showed signs of cerebral infarction (with reduced numbers of and disordered Nissl bodies and unclear structure), increased GRP78 immunopositive cells, increased expression of mRNA ( <0.05), and increased expressions of caspase12 and caspase3 mRNA ( <0.05). Compared with the model group, EA and EA+VEGF decreased mNSS scores at day 7 and 14 ( <0.05), showing alleviated signs of cerebral infarction, increased GRP78 immunopositive cells ( <0.05), increased mRNA expression ( <0.05), and decreased caspase12 and caspase3 mRNA expressions ( <0.05). The most obvious changes were found in the EA+VEGF group ( <0.05). No significant changes were observed in the sham-operation group over time ( <0.05). In comparison, mNSS scores, the signs of cerebral infarction, and the expressions of caspase12 and caspase3 decreased over time in the other groups ( <0.05), accompanied with increased GRP78 immunopositive cells and the expression of gene ( <0.05).

CONCLUSION

Electroacupuncture and intracerebral injection of VEGF promote tissue repair of rats with cerebral ischemic injury, possibly through down-regulating the expressions of caspase12 and caspase3 genes and up-regulating the expression of gene. The effect of electroacupuncture in combination with intracerebral injection of VEGF is superior to that of the single use of electroacupuncture.

摘要

目的

探讨电针(EA)及脑室内注射血管内皮生长因子(VEGF)对脑缺血再灌注损伤大鼠半胱天冬酶12(caspase12)、半胱天冬酶3(caspase3)及葡萄糖调节蛋白78kD(GRP78)基因的影响。

方法

将60只SD大鼠随机分为假手术组、模型组、电针组和电针+VEGF组,每组15只。采用大脑中动脉阻塞(MCAO)法建立脑缺血再灌注损伤模型。电针组和电针+VEGF组于造模后1天开始电针干预:每次30分钟,每天1次,共14天[穴位选择:百会(GV20)、曲池(LI11)、足三里(ST36)]。电针+VEGF组在首次电针后还于侧脑室内注射10μL VEGF165(0.025μg/μL)。分别于造模后第0天(造模后1天,电针干预前)、第7天和第14天对每组5只大鼠进行神经功能评分(mNSS)后处死。采用尼氏染色观察脑梗死灶的组织形态学变化。采用免疫组织化学法检测缺血脑组织中GRP78的活性。采用实时荧光定量聚合酶链反应(real-time PCR)检测缺血脑组织中caspase12、caspase3及GRP78 mRNA的表达。

结果

与假手术组比较,模型组大鼠mNSS评分升高(P<0.05),出现脑梗死征象(尼氏体数量减少、排列紊乱、结构不清),GRP78免疫阳性细胞增多,GRP78 mRNA表达升高(P<0.05),caspase12和caspase3 mRNA表达升高(P<0.05)。与模型组比较,电针组和电针+VEGF组在第7天和第14天mNSS评分降低(P<0.05),脑梗死征象减轻,GRP78免疫阳性细胞增多(P<0.05),GRP78 mRNA表达升高(P<0.05),caspase12和caspase3 mRNA表达降低(P<0.05)。电针+VEGF组变化最为明显(P<0.05)。假手术组各时间点比较差异无统计学意义(P>0.05)。与电针+VEGF组比较,其他组mNSS评分、脑梗死征象及caspase12和caspase3表达随时间降低(P<0.05),GRP78免疫阳性细胞及GRP78基因表达随时间升高(P<0.05)。

结论

电针及脑室内注射VEGF可促进脑缺血损伤大鼠的组织修复,可能是通过下调caspase12和caspase3基因表达、上调GRP78基因表达实现的。电针联合脑室内注射VEGF的效果优于单纯电针治疗。

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