mTOR/自噬通路在间歇性低氧全脑缺血再灌注大鼠海马区的表达及意义

Expression of mTOR/autophagy pathway in the hippocampus following cerebral ischemia/reperfusion injure in intermittent hypoxia rats

目的 比较常态大鼠雷帕霉素靶蛋白（mTOR）和间歇性低氧大鼠全脑缺血再灌注后海马区beclin1表达的变化,探讨mTOR/自噬通路在间歇性低氧加重脑缺血再灌注神经损伤中的作用.方法 100只雄性Wistar大鼠采用数字随机法随机分成假手术组（SO组,20只）、间歇性低氧组（IH组,20只）、单纯脑缺血再灌注组（I/R组,20只）、间歇性低氧脑缺血再灌注组（IH＋ I/R组,20只）、间歇性低氧脑缺血再灌注＋ mTOR抑制剂组（抑制剂组,20只）.造模前,IH组、IH＋ I/R组、抑制剂组给予间歇性低氧21 d.采用改良的Pulsinelli四血管阻断法制备脑缺血再灌注模型,分别于术后6、24 h,HE染色观察海马区神经细胞形态变化,免疫组织化学法、反转录聚合酶链反应（RT-PCR）检测大鼠脑组织海马区mTOR、beclin1蛋白表达情况.以SPSS 17.0统计软件进行统计学分析.结果 I/R组大鼠每个高倍镜视野中mTOR蛋白阳性细胞在6、24 h亚组分别为（22.38±0.46）、（24.16±0.60）个,与SO组的（14.65±0.48）、（15.40±0.58）个比较,差异有统计学意义（q值分别为90.59、106.83,P值均＜0.05）;beclin1蛋白阳性细胞分别为（8.58±0.58）、（10.58±0.49）个,与SO组的（2.06±0.23）、（2.10±0.30）个比较,差异有统计学意义（q值分别为95.88、119.44,P值均＜0.05）.IH组大鼠与SO组比较,mTOR、beclin1蛋白在各时间点表达增强（q值分别为32.94、47.31、63.68、78.45,P值均＜0.05）.与IH组比较,IH＋ I/R组大鼠在各时间点神经元结构损伤加重,mTOR、beclin1蛋白表达增强（q值分别为152.23、165.61、135.01、156.48,P值均＜0.05）.与I/R组比较,IH＋ I/R组大鼠在各时间点神经元结构损伤加重,mTOR、beclin1蛋白表达增强（q值分别为94.35、106.99、102.79、115.49,P值均＜0.05）.抑制剂组大鼠每个高倍镜视野中mTOR蛋白阳性细胞在6、24 h亚组分别为（26.60±0.37）、（28.51±0.52）个,与IH＋I/R组的（30.40±0.43）、（32.86±0.50）个比较,差异有统计学意义（q值分别为44.71、53.05,P值均＜0.05）;beclin1蛋白阳性细胞数在抑制剂组分别为（21.74±0.51）（24.32±0.49）个,在IH＋ I/R组分别为（15.57±0.57）、（18.78±0.43）个,差异有统计学意义（q值分别为90.74、78.03,P值均＜0.05）.结论 间歇性低氧可通过活化mTOR/自噬通路加重全脑缺血再灌注损伤后的神经损伤.

Objective To compare the changes in the expression of mTOR and beclin1 in the hippocampus of normal rats and intermittent hypoxia rats with cerebral ischemia/reperfusion,so as to explore the roles of mTOR/autophagy pathway in global cerebral ischemia/reperfusion injure aggravated by intermittent hypoxia.Methods One hundred healthy male Wistar rats were randomly divided into：sham operation group （SO group,n =20）,intermittent hypoxia group （IH group,n =20）,merely ischemia/ reperfusion group （I/R group,n =20）,intermittent hypoxia ischemia/reperfusion group（IH ＋ I/R group,n =20）,intermittent hypoxia ischemia/reperfusion ＋ mTOR inhibitor group （Inhibitor group,n =20）.IH group,IH ＋ I/R group and inhibitor group were respectively given intermittent hypoxia for 21 days before ischemia/ reperfusion.Ischemia animals were prepared cerebral ischemia-reperfusion model by improved pulsinelli four vessels block （4-VO）,the morphological changes of hippocampus nerve cells of rat brain were detected with HE respectively 6,24 h after ischemia,and the expressions of mTOR protein and beclin1 protein in hippocampus of rat brain was detected with immunohistochemistry and RT-PCR respectively 6,24 h after ischemia.SPSS 17.0 software was used to analyze the data.Results Compared with the SO group,the IH group increased the never cells morphology damages and the empression of mTOR and beclin1 （q value was 32.94,47.31,63.68,78.45,all P 〈 0.05）;the I/R group increased the never cells morphology damages and the empression of mTOR and beclin1 （mTOR in I/R group：22.38 ±0.46,24.16 ±0.60;mTOR in SO group：14.65 ± 0.48,15.40 ± 0.58;beclin1 in I/R group：8.58 ± 0.58,10.58 ± 0.49;beclin1 in SO group：2.06 ±0.23,2.10 ±0.30;the differences were significant,q value was 90.59,106.83,95.88,119.44,all P 〈0.05）.Compared with the IH group,IH ＋ I/R group increased the never cells morphology damages and the empression of mTOR and beclin1 （q value was 152.23,165.61,135.01,156.48,all P 〈 0.05）.Compared with the I/R group,IH ＋ I/R group increased the never cells morphology damages and the empression of mTOR and beclin1 （q value was 94.35,106.99,102.79,115.49,all P 〈0.05）.Compared with the IH ＋ I/R group,the inhibitor group decreased the never cells morphology damages and the expression of mTOR,increased the expression of beclin1 （mTOR in IH ＋ I/R group：30.40 ±0.43,32.86 ±0.50;mTOR in inhibitor group：26.60 ±0.37,28.51 ±0.52;beclin1 in IH ＋ I/R group：15.57 ± 0.57,18.78 ± 0.43;beclin1 in inhibitor group：21.74 ± 0.51,24.32 ± 0.49;the differences were significant,q value was 44.71,53.05,90.74,78.03,all P 〈 0.05）.Conclusion Intermittent hypoxia can aggravate the damage on nerve cells by activating mTOR/autophagy pathway after ischemia.