电针结合脑内注射血管内皮生长因子对脑缺血再灌注损伤大鼠内质网应激反应相关蛋白ATF 6、IRE 1、XBP 1、CHOP的影响

Electroacupuncture Combined with Intracerebral Injection of VEGF Improves Neurological Dysfunction Possibly by Down-regulating Expression of Endoplasmic Reticulum Stress Related Proteins ATF 6,etc.in Cerebral Ischemia-reperfusion Injury Rats

目的:观察电针及电针结合脑内注射血管内皮生长因子(VEGF)对脑缺血后再灌注损伤(CIRI)大鼠内质网应激反应(ERS)相关蛋白转录激活因子6(ATF 6)、肌醇需求酶1(IRE 1)、X盒结合蛋白1(XBP 1)、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)的影响,探讨电针结合脑内注射VEGF对CIRI的修复作用。方法:SD大鼠随机分为假手术组、模型组、电针组、VEGF组、电针+VEGF组,每组8只。采用线栓法制备CIRI模型。VEGF组及电针+VEGF组大鼠于造模完成24h后,向侧脑室内注入10!L VEGF(0.025!g/!L)。电针组及电针+VEGF组电针"百会"、左侧"曲池""足三里",1次/d,每次30min,共14d。对各组大鼠进行神经功能评分;尼氏染色法观察大鼠CIRI区域的组织形态学变化;Western blot法检测大鼠脑梗死区组织ERS相关蛋白ATF 6、IRE 1、XBP 1、CHOP的表达;荧光定量PCR法检测大鼠梗死区组织ATF 6、IRE 1、XBP 1、CHOP mRNA的表达。结果:与假手术组比较,模型组大鼠神经功能评分明显升高(P<0.05);与模型组比较,电针组、VEGF组、电针+VEGF组神经功能评分均明显降低(P<0.05);与电针组、VEGF组比较,电针+VEGF组神经功能评分明显降低(P<0.05)。与假手术组比较,模型组CIRI区域尼氏小体数明显减少(P<0.05);与模型组比较,电针组、VEGF组、电针+VEGF组CIRI区域尼氏小体数均明显增多(P<0.05);与电针组、VEGF组比较,电针+VEGF组尼氏小体数明显增多(P<0.05)。与假手术组比较,模型组大鼠CIRI区域ATF 6、IRE 1、XBP 1、CHOP蛋白及mRNA表达明显升高(P<0.05);与模型组比较,电针组、VEGF组、电针+VEGF组CIRI区域ATF 6、IRE 1、XBP 1、CHOP蛋白及mRNA表达均明显降低(P<0.05);与电针组、VEGF组比较,电针+VEGF组ATF 6、IRE 1、XBP 1、CHOP蛋白及mRNA表达明显降低(P<0.05)。结论:电针结合脑内注射VEGF可促进CIRI组织修复,其机制可能与下调ERS相关蛋白ATF 6、IRE 1、XBP 1、CHOP的表达有关,且其效果比单纯电针或单纯脑内注射VEGF更具优势。

Objective To observe the effect of electroacupuncture （EA） and EA combined with intracerebral injection of vascular endothelial growth factor （VEGF） on endoplasmic reticulum stress （ERS） related proteins and genes as activating transcription factor （ATF 6）, inositol requiring enzyme-1 （IRE 1）, CCAAT/enhancer binding protein homologous protein （CHOP）, X box-binding protein-1 （XBP 1） of cerebral ischemia reperfusion injury （CIRI） rats, so as to study its repair effect for CIRI. Methods Forty male SD rats were equally and randomly divided into 5 groups： sham operation, model, EA, VEGF and EA＋VEGF groups （n = 8）. The CIRI model was established by occlusion of the middle cerebral artery （MCAO） with thread embolism method. For rats of the sham operation group, the right common carotid artery was isolated without MCAO. EA （2 Hz/100 Hz, 1 -3 mA） was applied to ＂Baihui＂ （GV 20）, left ＂Quchi＂ （LI 11） and left ＂Zusanli＂ （ST 36） for 30 min, once a day for 14 days. For rats of the VEGF and EA＋ VEGF groups, 10 μL VEGF （0. 025 μg/μL） was injected into the lateral ventricle 24 h after successful modeling. The rats＇ neurological function was assessed by using the modified neurological severity score （mNSS）, and the his- topathological changes of cerebral tissue were observed by Nissl staining method. The expression levels of ERS related proteins and genes ATF 6, IRE 1, XBP 1 and CHOP were determined by western blot （WB） and fluorescent quantitative POR, separately. Results After modeling, the level of mNSS was significantly higher in the model group than in the sham operation group （ P〈0.05）, and the number of Nissl bodies was markedly lower in the model group than in the sham operation group （ P〈0.05）. Following the treatment, the mNSS was significantly lower in the EA, VEGF and EA ＋ VEGF groups than in the model group （P〈0.05）, and the numbers of Nissl bodies were obviously higher in the EA, VEGF and EA-I- VEGF groups than in the model group （ P〈0.05）, suggesting an improvement of neurological dysfunction and a repair of the injured cerebral tissue after the treat- ment. The levels of CIRI-induced increase of mNSS and CIRI-induced decrease of the number of Nissl bodies in the EA-I- VEGF group were respectively remarkably lower or higher than those of the simple EA and simple VEGF groups （ P〈0.05）. WB and PCR showed that the expression levels of ATF 6, IRE 1, XBP1 and CHOP proteins and genes were notably higher in the model group than in the sham operation group （P〈0.05）, and considerably lower in the EA, VEGF and EA-I-VEGF groups than in the model group （ P〈0.05）. Comparison among the three treatment groups showed that after the treatment, the expression levels of ATF 6, IRE 1, XBP1 and CHOP proteins and genes were obviously lower in the EA＋ VEGF group than in the EA and VEGF groups （P〈0.05）.Conclusion EA and EA plus intracerebral microinjection of VEGF can improve neurological function and promote cerebral tissue repair in CIRI rats, which is associated with their effects in down-regulating the expression of ERS related proteins ATF 6, IRE 1, XBP1 and CHOP. The effect of EA＋VEGF is superior to that of simple EA and simple VEGF.