富氢液对局灶性脑缺血-再灌注损伤大鼠线粒体通透性转换孔的影响

Effect of hydrogen-rich saline on mitochondrial permeability transition pore after focal cerebral ischemiareperfusion injury in rats

目的探讨富氢液对局灶性脑缺血-再灌注(I-R)损伤大鼠线粒体通透性转换孔(mPTP)的影响。方法 72只雄性SD大鼠随机均分为假手术组(S组)、I-R组、生理盐水组(NS组)、苍术苷组(A组)、富氢液组(H组)和富氢液+苍术苷组(HA组)。以线栓法制备大鼠大脑中动脉I-R损伤模型,缺血120min后再灌注。A组和HA组于再灌注前10min行侧脑室注射苍术苷15μl(3×10-5 mmol),NS组和H组侧脑室注射等容量生理盐水,其余组不注射生理盐水;H组和HA组于再灌注即刻腹腔注射富氢液5ml/kg,其余组腹腔注射等容量生理盐水。再灌注48h行神经功能缺陷评分后处死大鼠,取缺血侧顶叶皮层组织,分离细胞线粒体,测定mPTP活性、线粒体膜电位和细胞凋亡情况。结果与S组比较,其余各组再灌注48h神经功能缺陷评分均升高,mPTP活性升高,线粒体膜电位降低,TUNEL阳性细胞增多(P<0.05);与I-R组比较,H组和HA组神经功能缺陷评分降低,mPTP活性降低,线粒体膜电位升高,TUNEL阳性细胞减少(P<0.05);与H组比较,HA组神经功能缺陷评分升高,mPTP活性升高,线粒体膜电位降低,TUNEL阳性细胞增多(P<0.05)。结论富氢液通过抑制神经细胞mPTP开放、减少线粒体膜电位降低而减轻大鼠局灶性脑I-R损伤。

Objective To investigate the effect of hydrogen-rich saline on mitochondrial permeability transition pore（mPTP） after focal cerebral isehemia-reperfusion（I-R） injury in rats. Methods Seventy-two male SD rats were equally randomized into 6 groups of S, I-R, NS, A, H and HA. Focal cerebral PR injury rat model was induced by middle cerebral artery occlusion for 120 rain except those in group S. The rats in groups of A and HA received intraeerebroventricular injection with atractyloside 15 μl（3 × 10^-5mmol） at 10 min before reperfusion, while those in groups of NS and H received intracerebroventrieular injection with same volume of normal saline and those in ＂groups of S and LR did not receive intracerebroventrieular injection. Hydrogen-rich saline 5 ml/kg was injected intraperitoneally immediately after reperfusion in groups of H and HA, while the same volume of normal saline was injected in the other four groups. At 48 h after reperfusion, neurological deficit score （NDS） was used to evaluate the neurological function in rats. Then the rats were decapitated and the parietal cortex of the isehemie side was isolated. The opening degree of mPTP was assayed with spectrophotometry and the mitoehondrial membrane potential（MMP） was detected with Rhodamine 123 method. Cell apoptosis was analyzed by TUNEL assay. Results Compared with group S, NDS was increased, mPTP opening degree was enhanced, MMP was reduced and TUNEL positive cells were increased in the other five groups（P〈0. 05）. Compared with group I-R, NDS was decreased, mPTP opening degree was reduced, MMP was enhanced and TUNEL positive cells were decreased in groups of H and HA（P〈0. 05）. Compared with group H, NDS was increased,mPTP opening degree was enhanced, MMP was reduced and TUNEL positive cells were increased in group HA（P〈0. 05）. Conclusion Hydrogen-rich saline can attenuate focal cerebral I-R injury through inhibiting mPTP opening degree and MMP reduction.