不同药物联合预防泛影葡胺诱导急性肾损伤的机制研究

发布时间：2018-05-19 14:30

本文选题：对比剂诱导的急性肾损伤 + 内质网应激　；参考：《天津医科大学》2016年博士论文

【摘要】：研究背景及目的:对比剂诱导的急性肾损伤(contrast-induced acute kidney injury,CIAKI),又被称之为对比剂肾病(contrast induced nephropathy,CIN),是应用对比剂后发生的重要的临床并发症,它是行经皮冠状动脉介入治疗后继支架内血栓、支架内再狭窄后的第三大并发症。临床应用的对比剂主要包括泛影葡胺、碘佛醇、碘海醇等。CIAKI的发病机制十分繁杂。有研究表明,在CIAKI中肾小管损伤中,内质网应激而诱导的细胞凋亡发挥重要作用。已有研究推测,阿托伐他汀、普罗布考与前列地尔药物的抗氧化与抗炎或改善肾血管灌注作用可抑制肾小管细胞凋亡,均可能预防CIAKI的发生。此前已有研究显示阿托伐他汀可成功抑制内质网应激诱导的细胞凋亡作用从而预防CIAKI的发生。然而,该联合用药是否可通过抑制内质网应激诱导的细胞凋亡作用进而预防CIAKI的发生尚无具体研究。近几十年的研究发现许多可检测CIAKI的早期新型标记物,此前已有研究初步证明丛生蛋白可预测CIAKI的发生,但缺乏进一步的论证。因此,本研究拟用泛影葡胺建立大鼠CIAKI模型,通过不同药物联合干预,比较药物联合与阿托伐他汀单药对大鼠对比剂肾损伤的影响,检测各种药物联合是否通过抑制内质网应激特有分子伴侣进而探讨其可抑制内质网应激诱导的细胞凋亡从而发挥其对CIAKI后的肾功能的保护作用,并从药物联合角度进一步间接明确丛集素在CIAKI发生中的诊断意义及不同药物联合对其干预作用。研究方法:Wistar大鼠50只,随机分成5组:A组(阿托伐他汀组,n=10);P组(普罗布考+阿托伐他汀组,n=10);Q组(前列地尔+阿托伐他汀组,n=10);NC组(对比剂组,n=10);N组(对照组,n=10)。常温下适应性喂养7天后,1周末锁骨下静脉取血2ml测肾功能。予P组普罗布考灌胃14天后与阿托伐他汀同时灌胃10天。Q组与A组等体积0.5%羧甲基纤维素钠灌胃14天后,改用阿托伐他汀灌胃10天,N组与NC组每天等体积0.5%羧甲基纤维素钠溶液灌胃。灌胃21天后,第22天开始自锁骨下静脉注射泛影葡胺,N组注射生理盐水;Q组分别于注射对比剂前24小时、术前30分钟、术后24小时锁骨下静脉注射前列地尔,其他组等剂量生理盐水锁骨下静脉注射,术后继续灌胃直至处死。分别于注射对比剂后48小时及72小时取血2ml测肾功能,72小时后处死大鼠,取出肾脏,左肾用福尔马林固定,石蜡包埋送往病理科。右肾置于-80度液氮冰箱冷冻保存用于western blot及real-time RT-PCR实验。肾脏病理切片HE染色法及电镜下检测肾脏病理变化,用TUNEL染色法检测肾脏细胞凋亡。western blot及免疫组化检测内质网应激靶点GRP78、Clusterin、GADD153/CHOP、Caspase-12在蛋白水平上的表达,real-time RT-PCR检测上述靶点在核酸水平的表达。统计分析应用SPSS19.0统计软件。以均值±标准差(x±s)衡量计量资料数值,配对样本t检验方法比较组内数据;单因素方差分析法、Mann-Whitney U检验或Kruksal-Wallis检验比较组间数据。P㩳0.05表示差异有统计学意义。研究结果:1、泛影葡胺能引起大鼠肾功能肌酐指标明显增高,NC组造模前后差异值最大。阿托伐他汀组及联合普罗布考或前列地尔组肌酐升高值较对比剂组明显降低,联合用药组与空白组无明显差异,其中Q组对肾保护作用明显好于阿托伐他汀单药组。2、NC组GRP78、GADD153/CHOP及Caspase-12核酸表达水平最高,其中在GADD153/CHOP及Caspase-12的表达与其余四组相比有明显统计学差异;而对于联合用药及单药组之间的比较无明显统计学差异。3、Western blot实验中NC组GRP78、GADD153及Caspase-12蛋白表达量较其他四组显著升高,A组在GRP78靶点蛋白表达量明显高于P组,在GADD153明显高于联合用药组,在Caspase-12高于Q组。在GADD153和Caspase-12的表达Q组蛋白表达量明显低于P组,联合用药组在以上靶点的表达接近于N组甚至低于N组。4、在病理HE染色及电镜中,NC组中肾小管上皮细胞空泡样改变,部分上皮细胞坏死、基底膜增厚,肾小管腔狭窄,内质网及线粒体数量减少、发生肿胀,管腔面微绒毛杂乱脱落;而阿托伐他汀组及联合用药组肾小管损伤程度较对比剂组减轻,联合用药组病例损伤程度低于阿托伐他汀组。5、凋亡指数:NC组A组Q组P组N组,差异有统计学意义,其中A组较联合用药组显著升高。P组与Q组无明显差异。6、免疫组化:NC组GRP78、GADD153、Caspase-12表达量最高,A组在GRP78的表达上略高于联合用药组,无明显统计学差异,在GADD153的表达上高于联合用药组,与Q组有明显差异,在Caspase-12表达上与P组和Q组有明显差异。7、丛集素核酸表达五组间未见明显差异。NC组在蛋白表达水平明显高于其他四组,A组也显著高于联合用药组,Q组低于P组的表达。研究结论:1、内质网应激诱导的细胞凋亡通路在泛影葡胺诱导的急性肾损伤的发病中发挥了作用;阿托伐他汀、普罗布考及前列地尔对大鼠CIAKI的发病起到预防作用,这种作用可能是通过抑制内质网应激诱导的细胞凋亡通路而实现的,其中普罗布考、前列地尔与阿托伐他汀联合对对比剂肾损伤的肾保护作用可能优于阿托伐他汀组,前列地尔与阿托伐他汀的效果更明显。2、进一步阐明了,丛生蛋白是可以早期预测泛影葡胺诱导的急性肾损伤发生的新型生物标志物,普罗布考及前列地尔与阿托伐他汀的联合作用使CIAKI中丛生蛋白的表达下调证明其对泛影葡胺诱导的急性肾损伤后肾保护作用强于阿托伐他汀,其中前列地尔联合阿托伐他汀效果更显著。
[Abstract]:Research background and purpose: contrast-induced acute kidney injury (CIAKI) induced by contrast agent, which is also known as contrast agent nephropathy (contrast induced nephropathy, CIN), is an important clinical complication after using contrast agent. It is followed by percutaneous coronary intervention after stent thrombosis and stent restenosis. The third major postoperative complications. Clinical use of contrast agents mainly including meglumine, iodifol, iodiprol and other.CIAKI mechanisms are very complicated. Studies have shown that endoplasmic reticulum stress and induced apoptosis play an important role in CIAKI renal tubule injury. Antioxidation and anti-inflammatory or improved renal vascular perfusion can inhibit the apoptosis of renal tubular cells and may prevent the occurrence of CIAKI. Previous studies have shown that atorvastatin could successfully inhibit the apoptosis induced by endoplasmic reticulum stress and prevent the occurrence of CIAKI. However, whether the combined use of the combined drug can inhibit endoplasmic reticulum stress induced stress. There is no specific study on the role of apoptosis to prevent the occurrence of CIAKI. In recent decades, many early new markers to detect CIAKI have been found. Previous studies have preliminarily demonstrated that the cluster protein can predict the occurrence of CIAKI, but lack of further demonstration. Therefore, this study is to establish a rat CIAKI model by using meglumine. The effects of drug combination and atorvastatin on the renal injury of rat contrast agent were compared, and the effects of various drugs on the inhibition of endoplasmic reticulum stress specific molecular chaperone and the inhibition of the apoptosis induced by endoplasmic reticulum stress were explored to protect the renal function after CIAKI. The combined angle of drugs was used to further clarify the diagnostic significance of cluster in the occurrence of CIAKI and the effect of the combination of different drugs. Research methods: 50 Wistar rats were randomly divided into 5 groups: A group (atorvastatin group, n=10); P group (probucol + atorvastatin group, n= 10); Q group (alprostadil + atorvastatin group, n=10); group NC (contrast agent) Group, n=10, group N (control group, n=10). After 7 days of adaptive feeding at normal temperature, the function of 2ml was measured by the subclavian vein for the 1 weekend. In group P, 14 days after filling the stomach with atorvastatin for 10 days at the same time, group.Q and A group 0.5% carboxymethyl cellulose sodium for 14 days, with atorvastatin for 10 days, N group and NC group per day 0.5. 21 days after gavage, the subclavicular vein was injected into the subosseous vein for 21 days and the saline was injected into the subosseous vein on the twenty-second day. Group Q was injected with the contrast agent 24 hours before the injection, 30 minutes before the operation, 24 hours after the operation, and the other groups were injected with the subclavian vein, and the other groups were injected with the subclavian vein, and the gastric perfusion continued after the operation. After 48 hours and 72 hours after injection of contrast agent, the renal function was measured by 2ml. After 72 hours, rats were killed and kidney was taken out. The left kidney was fixed with formalin and paraffin was buried in the disease science. The right kidney was stored in -80 degree liquid nitrogen refrigerator for cryopreservation for western blot and real-time RT-PCR experiment. Renal pathological section HE staining and electron microscopy To detect renal pathological changes, TUNEL staining was used to detect.Western blot of renal cell apoptosis and the expression of GRP78, Clusterin, GADD153/CHOP, Caspase-12 at protein level in endoplasmic reticulum stress target, and real-time RT-PCR was used to detect the expression of the target at the level of nucleic acid. Statistical analysis applied SPSS19.0 software. Standard deviation (x + s) measure measurement data, paired sample t test method to compare intra group data; single factor variance analysis, Mann-Whitney U test or Kruksal-Wallis test comparison group data.P? 0.05 showed significant difference. 1, the results of the study were that the index of renal functional creatinine increased significantly in rats, and the model of NC group was created. The creatinine elevation value of atorvastatin group and combined proponol group or prostadil group was significantly lower than that of the contrast group. There was no significant difference between the combination group and the blank group. The protective effect of the Q group was better than that of the Atorvastatin single drug group.2, and the GRP78, GADD153/CHOP and Caspase-12 nucleic acids in the group NC were the highest. There was significant difference in the expression of GADD153/CHOP and Caspase-12 compared with the other four groups, but there was no significant difference between the combination and the single drug group.3. The expression of GRP78, GADD153 and Caspase-12 protein in group NC was significantly higher than that of the other four groups in the Western blot experiment, and the expression of the A group in GRP78 target protein was significantly higher than that of P. The expression of Q histone expression in GADD153 and Caspase-12 was significantly lower than that of group P in the group of GADD153. The expression of histone in GADD153 and Caspase-12 was significantly lower than that in group P. The expression of the combined drug group at the above target was close to that of the N group and even lower than the N group.4. In the pathological HE staining and the electrical mirror, the vacuoles like changes in the renal tubular epithelial cells in the NC group and some epithelial cells in the NC group. Necrosis, thickening of basal membrane, narrowing of renal tubule, decreased number of endoplasmic reticulum and mitochondria, swelling and tumbling of microvilli in the lumen, and reduction of renal tubule injury in atorvastatin group and combined drug group compared with contrast agent group, the degree of injury in the combined drug group was lower than that of atorvastatin group.5, apoptosis index: group Q P group N of group A group of group NC, group N, There was significant difference in the difference between group A and group Q. There was no significant difference between group.P and group Q,.6, immunohistochemistry: the expression of GRP78, GADD153, Caspase-12 in NC group was the highest, and the expression of A group in GRP78 was slightly higher than that of the combined drug group, and there was no significant difference in the expression of GADD153. The expression of spase-12 was significantly different from that of the P group and the Q group. There was no significant difference between the five groups. The expression level of the group.NC was significantly higher than that of the other four groups, and the A group was also significantly higher than the combination group, and the Q group was lower than the P group. 1, the apoptosis pathway induced by endoplasmic reticulum stress was in the acute kidney induced by meglumine. It plays a role in the pathogenesis of injury; atorvastatin, probucol and alprostadil play a preventive role in the pathogenesis of CIAKI in rats. This effect may be achieved by inhibiting the apoptosis pathway induced by endoplasmic reticulum stress, including propriopol, alprostadil and atorvastatin in the renal protection of contrastive renal injury. The effect of alprostadil and atorvastatin is more obvious than the Atorvastatin group, and the effect of alprostadil and atorvastatin is more obvious.2, further clarifying that the cluster protein is a new biomarker for the early prediction of acute renal injury induced by meglumine. The combination of probucol and alprostadil and atorvastatin makes the expression of CIAKI clump protein. The protective effect of alprostadil combined with atorvastatin was stronger than that of atorvastatin after kidney injury induced by meglumine dihydrochloride.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692

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