异氟烷后处理减轻大鼠局灶性脑缺血再灌注损伤时TGF-β3/Smad3信号通路与神经元凋亡的关系

Relationship between TGF-β3/Smad3 signaling pathway and neuronal apoptosis during reduction of focal cerebral ischemia-reperfusion injury by isoflurane postconditioning in rats

目的评价异氟烷后处理减轻大鼠局灶性脑缺血再灌注损伤时转化生长因子β3(TGF-β3)/果蝇MAD类似基因3(Smad3)信号通路与神经元凋亡的关系。方法清洁级健康雄性SD大鼠60只,6~8周龄,体重210~230 g,采用随机数字表法分为5组(n=12):假手术组(S组)、脑缺血再灌注组(I/R组)、脑缺血再灌注+异氟烷后处理组(I/R+ISO组)、脑缺血再灌注+吡非尼酮组(I/R+P组)和脑缺血再灌注+异氟烷后处理+吡非尼酮组(I/R+ISO+P组)。采用阻塞大脑中动脉1.5 h、再灌注24 h的方法制备大鼠局灶性脑缺血再灌注损伤模型。I/R+P组及I/R+ISO+P组于缺血前30 min时侧脑室注射TGF-β3抑制剂吡非尼酮5μg/kg;I/R+ISO组及I/R+ISO+P组于再灌注即刻吸入1.5%异氟烷1 h。再灌注24 h时,行神经功能缺损评分,然后处死大鼠,取脑组织,采用尼氏染色法测定神经元损伤率,采用TUNEL法测定神经元凋亡率,采用免疫荧光法测定TGF-β3表达,采用Western blot法检测TGF-β3、磷酸化Smad3(p-Smad3)、caspase-3、Bax和Bcl-2的表达水平。结果与S组比较,I/R组神经功能缺损评分、神经元损伤率和神经元凋亡率升高,脑组织TGF-β3、caspase-3和Bax表达上调,p-Smad3和Bcl-2表达下调(P<0.05);与I/R组比较,I/R+ISO组神经功能缺损评分、神经元损伤率和神经元凋亡率降低,脑组织TGF-β3、p-Smad3和Bcl-2表达上调,caspase-3和Bax表达下调,I/R+P组神经功能缺损评分、神经元损伤率和神经元凋亡率升高,脑组织TGF-β3和p-Smad3表达下调,caspase-3表达上调(P<0.05);与I/R+ISO组比较,I/R+ISO+P组神经功能缺损评分、神经元损伤率和神经元凋亡率升高,脑组织TGF-β3、p-Smad3和Bcl-2表达下调,caspase-3和Bax表达上调(P<0.05)。结论异氟烷后处理可通过激活TGF-β3/Smad3信号通路,抑制神经元凋亡,减轻大鼠局灶性脑缺血再灌注损伤。

Objective To evaluate the relationship between transforming growth factor beta-3(TGF-β3)/mammalian homologs of the drosophila mad gene 3(Smad3)signaling pathway and neuronal apoptosis during reduction of focal cerebral ischemia-reperfusion(I/R)injury by isoflurane postconditioning(ISO)in rats.Methods Sixty clean-grade healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 210-230 g,were randomly divided into 5 groups(n=12 each):sham operation group(group S),cerebral I/R group(group I/R),cerebral I/R plus isoflurane postconditioning group(group I/R+ISO),cerebral I/R plus pirfenidone group(group I/R+P),and cerebral I/R plus isoflurane postconditioning plus pirfenidone group(group I/R+ISO+P).Local cerebral I/R was produced by middle cerebral artery occlusion for 1.5 h followed by 24-h reperfusion in anesthetized rats.Pirfenidone 5μg/kg was injected into the lateral ventricle at 30 min before ischemia in I/R+P and I/R+ISO+P groups.In I/R+ISO and I/R+ISO+P groups,1.5%isoflurane was inhaled for 1 h starting from the time point immediately after onset of reperfusion.Neuro-functional deficit was assessed using neurologic deficit scores(NDS)at the end of reperfusion.Then the animals were sacrificed,and brain tissues were removed for determination of the neuronal damage rate(by Nissl staining),neuronal apoptosis rate(by TUNEL),expression of TGF-β3(using immunofluorescence),and expression of TGF-β3,phosphorylated Smad3(p-Smad3),caspase-3,Bax and Bcl-2(by Western blot).Results Compared with group S,the NDS,neuronal damage rate and apoptosis rate of neurons were significantly increased,the expression of TGF-β3,caspase-3 and Bax was up-regulated,and the expression of p-Smad3 and Bcl-2 was down-regulated in group I/R(P<0.05).Compared with group I/R,the NDS,neuronal damage rate and apoptosis rate of neurons were significantly decreased,the expression of TGF-β3,p-Smad3 and Bcl-2 was up-regulated,and the expression of caspase-3 and Bax was down-regulated in group I/R+ISO,and the NDS,neuronal damage rate and apoptosis rate of neurons were significantly increased,the expression of TGF-β3 and p-Smad3 was down-regulated,and the expression of caspase-3 was up-regulated in group I/R+P(P<0.05).Compared with group I/R+ISO,the NDS,neuronal damage rate and apoptosis rate of neurons were significantly increased,the expression of TGF-β3,p-Smad3 and Bcl-2 was down-regulated,and the expression of caspase-3 and Bax was up-regulated in group I/R+ISO+P(P<0.05).Conclusion Isoflurane postconditioning can inhibit neuronal apoptosis by activating the TGF-β3/Smad3 signaling pathway,thus reducing focal I/R injury in rats.