芹黄素联合缺血后处理对大鼠肾缺血再灌注损伤的影响

2017-07-05 23:04刘洋刘修恒王磊
中国医药导报 2017年15期
关键词:凋亡

刘洋 刘修恒 王磊

[摘要] 目的 探討芹黄素联合缺血后处理对大鼠肾缺血再灌注损伤的影响。 方法 将50只大鼠随机分为5组:假手术组(sham组)、缺血再灌注组(I/R组)、芹黄素组(api组)、缺血后处理组(IPO组)、芹黄素+缺血后处理组(api+IPO组)。建立大鼠肾缺血再灌注损伤模型,处死动物取肾组织石蜡包埋切片,观察肾组织病理学变化,免疫组化检测Fas、Caspase-3和Bcl-2表达,Real-time PCR检测Fas、Caspase-3、PARP-1和Bcl-2表达水平,Western blot检测Caspase-3、Bax和Bcl-2表达水平。 结果 与sham组相比,I/R组病理组织显示肾小管损伤严重;免疫组化示Fas、Caspase-3蛋白表达增强,Bcl-2表达降低(P < 0.01);Real-time PCR示Fas、Caspase-3和PARP-1 mRNA表达明显上调,Bcl-2 mRNA表达明显下调(P < 0.01);Western blot示Caspase-3和Bax表达水平明显升高,Bcl-2表达水平明显降低,差异有高度统计学意义(P < 0.01)。与I/R组相比,api组、IPO组和api+IPO组病理组织显示肾小管损伤显著改善;免疫组化示Fas、Caspase-3蛋白表达下调,Bcl-2表达上调(P < 0.05);Real-time PCR示Fas、Caspase-3和PARP-1 mRNA表达明显降低,Bcl-2 mRNA表达明显增高(P < 0.05);Western blot示Caspase-3和Bax表达水平明显下降,Bcl-2表达水平明显升高,差异有统计学意义(P < 0.05)。 结论 芹黄素和缺血后处理均能减轻肾脏缺血再灌注损伤,其机制可能与其上调Bcl-2基因和下调Bax、Caspase-3等基因表达有关,从而抑制肾缺血再灌注损伤后所造成的细胞凋亡。

[关键词] 缺血再灌注;芹黄素;缺血后处理;凋亡

[中图分类号] R364.1 [文献标识码] A [文章编号] 1673-7210(2017)05(c)-0022-04

[Abstract] Objective To investigate the effects of apigenin combined with ischemic postconditioning on renal ischemia/reperfusion injury in rats. Methods Fifty rats were randomly divided into 5 groups: sham-operated group (sham group), ischemia/reperfusion group (I/R group), apigenin group (api group), ischemic postconditioning group (IPO group) and api+IPO group. The rat model with renal ischemia/reperfusion injury was established, then the animals were sacrificed and nephridial tissue was taken to make paraffin-embedded sections. The pathologic changes of nephridial tissue were observed, the expression of Fas, Caspase-3, Bax were detected by immunohistochemistry, the expression of Fas, Caspase-3, PARP-1 and Bcl-2 were detected by real-time PCR, the expression of Caspase-3, Bax and Bcl-2 were detected by Western blot. Results Compared with the sham group, in I/R group, the pathological tissue showed that kidney tubules were damaged seriously; the immunohistochemistry showed that the expression of Fas and Caspase-3 were enhanced, the expression of Bcl-2 was decreased (P < 0.01); Real-time PCR showed that the expression of Fas, Caspase-3 and PARP-1 mRNA were up-regulated significantly, the expression of Bcl-2 was down-regulated significantly (P < 0.01); Western blot showed that the expression of Caspase-3 and Bax were increased significantly, the expression of Bcl-2 was reduced significantly, the differences were highly statistically significant (P < 0.01). Compared with I/R group, in api+I/R group, IPO group and api+IPO group, the pathological tissue showed that kidney tubules were alleviated; the immunohistochemistry showed that the expression of Fas and Caspase-3 were reduced, the expression of Bcl-2 was increased (P < 0.05); Real-time PCR showed that the expression of Fas, Caspase-3 and PARP-1 mRNA were down-regulated significantly, the expression of Bcl-2 was up-regulated significantly (P < 0.05); Western blot showed that the expression of Caspase-3 and Bax were reduced significantly, the expression of Bcl-2 was increased significantly, the differences were statistically significant (P < 0.05). Conclusion Both apigenin and ischemic postconditioning can reduce the renal ischemia/reperfusion injury, the mechanism may be related to up-regulating the expression of Bcl-2 and down-regulating the expression of Bax, Caspase-3, so as to inhibit the cell apoptosis caused by renal ischemia/reperfusion injury.

[Key words] Ischemia/reperfusion; Apigenin; Ischemic postconditioning; Apoptosis

肾缺血再灌注损伤是指肾组织缺血时和重获血流灌注或氧供应后,器官和组织发生损伤甚至功能衰竭[1]。肾缺血再灌注损伤常常发生在严重创伤、急性失血、中毒性休克和肾移植术后,极易引发急性肾衰竭[2]。缺血后处理是指组织或器官在长期缺血后再重获多次血流灌注或氧供应的过程,过程中缺血组织和器官的内源性保护机制被启动,缓解组织和器官的缺血再灌注损伤[3]。缺血后处理能抑制肾缺血再灌注损伤诱发的肾组织细胞凋亡,但机制尚不十分清楚[4-5]。芹黄素是一种天然多酚类化合物,它以黄色素的形式广泛存在于多种植物中,能产生明显的抗氧化作用,可清除体内过多的自由基,稳定细胞膜结构[6-7]。本研究旨在探讨芹黄素联合缺血后处理方案对肾缺血再灌注损伤的相关保护作用机制。

1 材料与方法

1.1 材料

1.1.1 实验动物 3月龄左右健康雄性SD大鼠50只,体重为220~300 g,购于湖北省疾病预防控制中心(合格证号:42000600014826),饲养于武汉大学人民医院实验动物中心清洁级动物室。

1.1.2 实验材料 芹黄素,纯度为98%,购于上海阿拉丁试剂有限公司(货号A106676)。考马斯亮蓝蛋白测定试剂盒、Fas、Caspase-3、PARP、Bcl-2抗体均购于SantaCruz公司,BCA蛋白浓度测定试剂盒购于碧云天生物技术研究所。

1.2 方法

1.2.1 模型的建立与分组 所有的动物实验操作均依照武汉大学人民医院实验动物委员会规定的协议及相关推荐进行。大鼠被随机分为以下5组:①假手术组(sham组)(n = 10):以1%浓度的戊巴比妥钠溶液按照50 mg/kg的剂量行腹腔注射麻醉,暴露两侧肾脏后,分离肾包膜及肾动静脉,切除右腎后缝合腹壁;②缺血再灌注组(I/R组)(n = 10):暴露两侧肾脏后,分离肾包膜及肾动静脉,切除右肾后使用无损伤血管夹夹闭左肾肾动脉和肾静脉,待肾脏颜色变为暗红色即可确定左肾血流被阻断,45 min后松开动脉夹,肾脏表面颜色恢复为鲜红色,可认为血流恢复正常,逐层关闭腹腔;③芹黄素组(api组)(n = 10):手术方法与I/R组保持一致,但在术前30 min腹腔注射10 mg/kg的芹黄素;④缺血后处理组(IPO组)(n = 10):暴露两侧肾脏后,分离肾包膜及肾动静脉,切除右肾后使用无损伤血管夹夹闭左肾肾动脉和肾静脉,在阻断左肾动脉血流45 min后,给予反复3次的恢复血流10 s,阻断血流10 s的处理,然后再完全恢复血流供应,逐层关闭腹腔;⑤芹黄素+缺血后处理组(api+IPO组)(n = 10):手术方法与IPO组一致,但在术前30 min腹腔注射10 mg/kg的芹黄素。

1.2.2 肾组织病理学检查 石蜡切片行HE染色,置于日本Olympus显微镜下观察其病理改变并拍摄。

1.2.3 免疫组织化学检测 肾组织石蜡切片,厚4 μm,按SP法免疫组化试剂盒进行操作,加入Fas、Caspase-3和Bcl-2(1∶100)抗体及相应二抗,DAB显色5 min后封片。细胞浆或细胞核有棕黄色表达者为阳性细胞。

1.2.4 Real-time PCR检测 用Trizol提取总RNA,琼脂糖电泳检测RNA完整性,分光光度计检测RNA浓度和纯度。取总RNA 1 μg,逆转录生成cDNA。以β-actin为内参照,分析基因的相对表达量。

1.2.5 Western blot检测蛋白表达 肾组织剪碎匀浆加入裂解液提取蛋白,冰上放置40 min,离心取上清,采用BCA试剂盒检测蛋白浓度。取20 μg蛋白样品上样,于10%聚丙烯酰胺凝胶上进行电泳,电泳结束后100 V电转印至PVDF膜上。50 g/L脱脂牛奶封闭2 h,稀释一抗4℃孵育过夜。之后将辣根过氧化物酶(HRP)标记的二抗1∶2000稀释后加入孵育,洗膜后ECL法显色并照相。

1.3 统计学方法

采用SPSS 17.0统计软件进行数据处理。计量资料采用均数±标准差(x±s)表示,多组比较采用方差分析,组间两两比较采用q检验。以P < 0.05为差异有统计学意义。

2 结果

2.1 组织病理学结果

HE病理染色结果示,sham组肾小管排列整齐,肾小管未见明显的形态学改变。I/R组肾小管上皮细胞肿胀,部分细胞明显变性坏死,肾小管明显扩张,管腔内可见细胞管型。与I/R组相比,api+IPO组、api组和IPO组肾小管损伤程度明显减轻,有少量肾小管上皮细胞轻度水肿,管型少见,有少量炎性细胞浸润。见图1。

2.2 Fas、Caspase-3和Bcl-2免疫组织化学法结果

免疫组织化学法结果示,sham组Fas、Caspase-3蛋白表达较低,Bcl-2蛋白表达较高;I/R组Fas、Caspase-3表达明显上调,Bcl-2蛋白表达明显下调。然而经api或IPO处理后Fas和Caspase-3表达量显著降低,Bcl-2表达量明显增高(P < 0.05)。见图2(封三)。

2.3 Fas、Caspase-3、PARP-1和Bcl-2 mRNA水平比较

Real-time PCR结果显示,sham组大鼠肾组织中Fas、Caspase-3和PARP-1 mRNA表达较低,Bcl-2 mRNA表达较高。经过I/R后,肾组织中Fas、Caspase-3和PARP-1 mRNA表达明显上调(P < 0.01),Bcl-2 mRNA表达明显下调(P < 0.01)。与I/R组比较,api+IPO组、api组和IPO组Fas、Caspase-3和PARP-1 mRNA表达显著下降(P < 0.05),Bcl-2 mRNA表达显著升高(P < 0.05),其中api+IPO组表现更为明显,而IPO组和api组的mRNA表达水平差异无统计学意义(P > 0.05)。见图3。

2.4 各组大鼠Western blot结果

Western blot结果示,sham组Caspase-3和Bax表达水平较低,Bcl-2表达水平较高。I/R组Caspase-3和Bax表达水平明显升高,而Bcl-2表达水平明显降低(P < 0.01)。此外,api+IPO组、api组和IPO组的Caspase-3和Bax的表达水平相对I/R显著下调,Bcl-2表达水平显著上调(P < 0.05)。其中api+IPO组的趋势更为明显(P < 0.05)。见图4。

3 讨论

急性肾功能衰竭是临床上常见的危重疾病之一,肾缺血再灌注损伤是其中最主要的病因[8]。肾缺血再灌注损伤的机制研究早已成为热点,但尚未发现有效的治疗方法。Fas是TNF家族成员之一,在心脏、肾脏和肝脏中呈现高表达[9]。FasL是细胞表面的一种Ⅱ型膜蛋白,多表达于NK细胞和被激活的T淋巴细胞表面[10]。Fas和FasL结合过程中生成三聚体结构,并募集相关的凋亡蛋白,结合成死亡起始信号复合体,激活Caspase-3执行细胞死亡程序。活化的Caspase-3能够破坏细胞内的某些结构蛋白和聚腺苷二磷酸核糖聚合酶(PARP),导致细胞骨架解体,从而引起细胞凋亡[11-12]。本研究结果表明,芹黄素和缺血后处理均能下调肾缺血再灌注后Fas和Caspase-3的表达水平,而且其下调趋势在联合处理方案中更为明显。这说明芹黄素和缺血后处理都能够有效地抑制Caspase-3的表达,从而阻断Fas/FasL介导的凋亡途径。

Bcl-2基因是早年在滤泡性非霍奇金淋巴瘤细胞中分离出来的一种癌基因[13]。Bcl-2能够减少自由基的生成,抑制内质网内Ca2+的释放和线粒体膜的通透性,阻断Caspase-3的激活过程,从而抑制细胞凋亡[14-15]。Bax是启动细胞程序性死亡的关键基因,能促进细胞因子缺失而诱导细胞凋亡[16]。Bcl-2与Bax的比例决定了细胞对凋亡刺激的敏感性,影响细胞的存活或者凋亡状态[13]。本研究结果證实芹黄素和缺血后处理显著上调缺血再灌注后的大鼠肾组织中Bcl-2/Bax比值,抑制细胞凋亡。

不同于以往针对肾缺血再灌注损伤的单一治疗,本研究采用了芹黄素联合缺血后处理的方案。实验结果表明,联合方案更为有效地减轻缺血再灌注后肾脏组织的损伤,改善肾功能,增强细胞的抗氧化能力,下调Fas、Caspase-3、Bax等凋亡基因,上调Bcl-2基因,抑制细胞凋亡。芹黄素联合缺血后处理方案实施方法简单,效果强于单一处理,更具有直接的临床实用价值。

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(收稿日期:2017-01-21 本文編辑:张瑜杰)

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