DYYG化合物诱导足细胞自噬对PAN肾病的肾脏保护作用

发布时间：2018-06-09 03:07

本文选题：DYYG化合物 + 足细胞病　；参考：《吉林大学》2017年博士论文

【摘要】：背景与目的:足细胞损伤与肾小球疾病的发生发展密切相关,本实验采用的嘌呤霉素氨基核苷(PAN)诱导大鼠肾病模型(PAN肾病模型)与PAN体外诱导足细胞凋亡为两个经典的足细胞损伤模型,广泛用于足细胞损伤研究当中。由我们的合作方美国刘宏宇教授课题组研发的DYYG化合物(C16H12N8O.HCL)是甲基并咪唑-VA二唑-吡啶并咪唑的衍生物的盐酸盐,是一种新型小分子化合物,其分子量为368。我们前期研究发现DYYG化合物对于足细胞损伤模型具有显著修复作用,该论文主旨为探讨DYYG化合物是否通过PI3K/Akt/m TOR信号通路诱导足细胞自噬,产生对PAN肾病足细胞损伤的修复,从而对肾脏起保护作用。方法:第一部分:体外培养足细胞实验:分为CON组、DYYG组、PAN组、PAN+DYYG组,按分组条件培养24小时,Western blot、免疫荧光方法观察PAN诱导足细胞损伤后,DYYG对其标志蛋白表达的影响。流式细胞仪检测DYYG对PAN诱导足细胞凋亡的影响。第二部分:体外培养足细胞实验:分为CON组、DYYG组、PAN组、PAN+DYYG组、PAN+DYYG+3-MA组、3-MA组,按分组条件培养24小时,应用共聚焦显微镜观察DYYG对m RFP-GFP-LC3腺病毒转染足细胞自噬的影响,透射电镜下观察足细胞内自噬体数目的变化,Western blot检测足细胞LC3-Ⅱ、Beclin-1、P62的表达水平。第三部分:60只雄性SD大鼠随机分为CON组、PAN组,PAN+DYYG组,PAN+TAC组,每组15只,构建PAN肾病模型。于0、7、14、21、28天,分五批处死动物,12只/次,测每个时间点24小时尿量、尿蛋白定量、血Cr、血BUN。免疫组织化学方法检测DYYG对PAN肾病模型肾小球足细胞标志蛋白Podcin表达的影响。免疫荧光方法共聚焦显微镜下观察DYYG对PAN肾病模型肾小球足细胞特异性骨架蛋白Synaptopodin表达的影响。透射电镜下观察DYYG对PAN肾病模型肾小球足细胞病理结构的影响。第四部分:Western blot明确DYYG对PAN肾病模型足细胞LC3-Ⅱ、Beclin-1、P62以及PTEN、PI3K、Akt及其下游m TOR信号分子表达的影响。结果:第一部分:按分组条件培养足细胞24小时后,Western blot结果发现,与CON组相比,PAN组足细胞标志蛋白Synaptopodin、Nephrin、Podcin、ZO-1蛋白表达下调(均P0.05)。与PAN组相比,PAN+DYYG组足细胞标志蛋白Synaptopodin、Nephrin、Podcin、ZO-1蛋白表达上调(均P0.05),而DYYG组与CON组相比则无明显差异。免疫荧光方法结果显示,PAN组足细胞特异性骨架蛋白Synaptopodin荧光强度显著低于CON组,Synaptopodin丝状结构破坏,皱缩于核周表达。PAN+DYYG组足细胞特异性骨架蛋白Synaptopodin荧光强度显著高于PAN组。而DYYG组与CON组相比则无明显差异。流式细胞仪检测结果发现,与CON组相比,PAN组足细胞凋亡比例明显增加(均P0.05),与PAN组相比,PAN+DYYG组足细胞凋亡比例明显降低(均P0.05),而DYYG组与CON组相比则无明显差异。第二部分:应用共聚焦显微镜观察DYYG对m RFP-GFP-LC3腺病毒转染足细胞自噬的影响,结果显示,CON组足细胞内见红色斑点及黄色斑点表达。与CON组相比,PAN组足细胞红色斑点及黄色斑点聚集数量明显减少(P0.05)。与PAN组相比,PAN+DYYG组足细胞红色斑点及黄色斑点聚集数量明显增加(P0.05)。经3-MA预处理1小时后再给予PAN+DYYG,DYYG恢复足细胞自噬的作用减弱(P0.05)。DYYG组、3-MA组与CON组相比则无明显差异。透射电镜下观察足细胞内自噬体数量,结果显示,与CON组相比,PAN组足细胞自噬体数量明显减少(P0.05)。与PAN组相比,PAN+DYYG组足细胞自噬体数量明显增加(P0.05)。DYYG组、3-MA组与CON组相比则无明显差异。经3-MA预处理1小时后再给予PAN+DYYG,足细胞内自噬体数量无增加。Western blot检测足细胞自噬相关蛋白的表达水平均验证上述实验结果,表明DYYG可增强PAN诱导足细胞损伤模型的自噬水平,从而达到足细胞保护作用。第三部分:成功建立SD大鼠PAN肾病模型,第7天PAN组、PAN+DYYG组、PAN+TAC组与CON组相比大鼠体重未见明显变化,差异无统计学意义(均P0.05)。第14天、第21天及第28天CON组、PAN+DYYG组及PAN+TAC组与PAN组比较,大鼠体重逐渐增加,差异有统计学意义(均P0.05),PAN+DYYG组与PAN+TAC组比较差异无统计学意义(均P0.05)。第7天,PAN组、PAN+DYYG组、PAN+TAC组与CON组比较大鼠尿量明显减少,出现蛋白尿,血Cr及血BUN值明显升高(均P0.05)。第14天,PAN组、PAN+DYYG组、PAN+TAC组与CON组比较大鼠尿量明显减少,24小时尿蛋白定量、血Cr及血BUN值较第7天降低,而PAN+DYYG组、PAN+TAC组与PAN组比较,24小时尿蛋白定量、血Cr及血BUN值降低更明显(均p0.05)。第21天、第28天,PAN组、PAN+DYYG组、PAN+TAC组24小时尿蛋白定量、血Cr及血BUN浓度逐渐降低,与PAN组比较,PAN+DYYG组、PAN+TAC组降低更明显,24小时尿量明显增加,PAN+DYYG组与PAN+TAC组比较差异无统计学意义(均P0.05)。免疫组织化学方法检测PAN组足细胞标志蛋白Podcin表达显著低于CON组,PAN+DYYG组及PAN+TAC组足细胞标志蛋白Podcin表达显著高于PAN组,两者相比则无明显差异(P0.05)。免疫荧光方法共聚焦显微镜结果显示,PAN组足细胞特异性骨架蛋白Synaptopodin荧光强度显著低于CON组,PAN+DYYG组足细胞特异性骨架蛋白Synaptopodin荧光强度显著高于PAN组。而DYYG组与CON组相比则无明显差异。透射电镜结果显示,PAN注射第7天,PAN肾病模型组,足突融合,溶酶体坏死,肾间质水肿,PAN肾病给予DYYG、TAC治疗组,足突增宽,肿胀。PAN注射第14天,PAN肾病模型组,病变更加严重,足突广泛融合,部分足突消失,而给予DYYG、TAC治疗组,与第7天比较,足突形态好转,但两者未见明显差异。PAN注射第21天,PAN肾病组及给予DYYG、TAC治疗组,足突肿胀,融合好转,与PAN肾病模型组相比,给予DYYG、TAC治疗组好转更明显,与CON组足突结构相似,但两者未见明显差异。PAN注射第28天,给予DYYG、TAC治疗组足突形态结构恢复正常,但PAN肾病模型组足突宽度仍高于CON组。通过对足突超微结构的观察,与PAN肾病模型组相比,给予DYYG、TAC治疗组足突融合程度明显低于PAN肾病模型组,DYYG、TAC可以改善和逆转足突形态改变。第四部分:Western Blot方法结果显示:PAN组足细胞内PTEN蛋白表达量下调,PI3K、p Akt、m TOR表达量明显增加,LC3Ⅱ、Beclin表达下调;P62蛋白表达量增加,信号通路蛋白及自噬相关蛋白表达量与CON组相比,差异有统计学意义(均P0.05)。与PAN组比较,PAN+DYYG组足细胞PTEN表达上调,PI3K、p Akt、m TOR表达下调;LC3Ⅱ、Beclin表达上调;P62蛋白表达量减少,信号通路及自噬相关蛋白表达量与PAN组相比,差异有统计学意义(均P0.05)。而PAN+LY294002组,PI3K抑制剂LY294002可抑制PI3K、p Akt、m TOR表达,上调自噬相关蛋白LC3Ⅱ、Beclin,下调P62表达(均P0.05),但对PTEN表达差异无统计学意义(P0.05)。结论:本研究证实PAN可抑制足细胞自噬,诱导足细胞损伤。DYYG干预可减轻PAN诱导的足细胞自噬抑制,增强足细胞自噬,改善足细胞损伤,其保护机制可能是通过上调PTEN表达,抑制PI3K/Akt/m TOR信号通路活化而实现的。
[Abstract]:Background and objective: podocyte injury is closely related to the development of glomerular disease. The rat model of nephrosis induced by PAN (PAN nephropathy model) and PAN induced foot cell apoptosis in vitro are two classic foot cell damage models, which are widely used in the study of foot cell injury. The DYYG compound (C16H12N8O.HCL), developed by Professor Liu Hongyu in the United States, is a salt of methyl and imidazole -VA two azol-pyridine and imidazole derivatives. It is a new small molecular compound. Its molecular weight is 368.. Our previous study found that DYYG compounds have a significant repair effect on the model of foot cell damage. The main purpose of this paper is to explore the main purpose of this paper. Whether DYYG compounds can induce autophagy through PI3K/Akt/m TOR signaling pathway to repair foot cell injury in PAN nephrosis and protect the kidneys. Method: Part 1: in vitro cultivation of podocyte experiment: CON group, DYYG group, PAN group, PAN+DYYG group, 24 hours, Western blot, immunofluorescence method according to the group conditions. The effect of DYYG on the expression of its marker protein after PAN induced podocyte injury. Flow cytometry was used to detect the effect of DYYG on PAN induced podocyte apoptosis. The second part: in vitro cultivation of podocyte experiment: CON group, DYYG group, PAN group, PAN+DYYG group, PAN+DYYG+3-MA group, 3-MA group, 24 hours by group conditions, and confocal microscopy The effect of DYYG on autophagy of M RFP-GFP-LC3 adenovirus transfected foot cells was observed. Under transmission electron microscope, the number of autophagosomes in podocytes was observed. Western blot was used to detect the expression level of LC3- II, Beclin-1 and P62 in podonote. The third part: 60 male SD rats were randomly divided into CON group, PAN group, PAN+DYYG group, and 15 rats each. On 0,7,14,21,28 days, five batches of animals were killed, 12 / time, 24 hours per hour urine volume, urine protein quantitative, blood Cr, and blood BUN. immunohistochemical method were used to detect the effect of DYYG on the expression of glomerular podocyte marker protein Podcin in PAN nephropathy model. DYYG against PAN nephropathy model glomerular foot thin under the immunofluorescence method. Effect of the expression of cellular specific skeleton protein Synaptopodin. The effect of DYYG on the pathological structure of glomerular podocytes in PAN nephropathy model was observed under transmission electron microscope. Fourth part: Western blot clearly the effect of DYYG on the expression of LC3- II, Beclin-1, P62 and PTEN, PI3K, and the lower reaches of the PAN nephropathy model. Results: the first part Western blot results showed that the expression of podocyte protein Synaptopodin, Nephrin, Podcin, ZO-1 protein was down regulated (all P0.05) in the PAN group compared with the CON group. The results of immunofluorescence showed that the fluorescence intensity of the specific skeleton protein Synaptopodin of the PAN group was significantly lower than that of the CON group, and the Synaptopodin filamentous structure was destroyed, and the expression of the specific skeleton protein Synaptopodin fluorescence intensity of the.PAN+DYYG group was significantly higher than that of the PAN group, while the DYYG group and the CON group phase were significantly higher than that of the group of Synaptopodin. Compared with the CON group, flow cytometry showed that the percentage of apoptotic podocytes in the PAN group was significantly increased (P0.05). Compared with the PAN group, the percentage of apoptotic podthe cells in the PAN+DYYG group was significantly lower (P0.05), while the DYYG group was not significantly different from that in the CON group. Second part: a confocal microscope was used to observe DYYG to m RFP-GFP-LC. The effect of 3 adenovirus transfection on autophagy of foot cells showed that red spots and yellow spots were expressed in the foot cells of CON group. Compared with group CON, the number of red spots and yellow spots in the foot cells of group PAN decreased significantly (P0.05). Compared with the PAN group, the number of red spots and yellow spots in the PAN+DYYG group increased significantly (P0.05). Through 3-, the number of the podocytes in the group of PAN was significantly increased. MA was given to PAN+DYYG after 1 hours of preconditioning, and the effect of DYYG on autophagy decreased (P0.05) in.DYYG group, and there was no significant difference between 3-MA and CON groups. The number of autophagic bodies in the podocytes was observed under transmission electron microscopy. The results showed that the number of autophagic bodies in the PAN group was significantly reduced (P0.05) compared with the CON group (P0.05). The PAN+DYYG group was more thin than the PAN group. There was a significant increase in the number of autophagic bodies (P0.05).DYYG, and there was no significant difference between the 3-MA group and the CON group. PAN+DYYG was given after 1 hours of 3-MA pretreatment. The number of autophagic in the podocytes was not increased by.Western blot to detect the expression level of autophagy related protein in the podocyte. The results showed that DYYG could enhance the injury of the foot cell induced by PAN. The third part: the model of PAN nephropathy in SD rats was successfully established. Seventh days PAN group, PAN+DYYG group, PAN+TAC group and CON group had no significant changes in weight (P0.05). Fourteenth days, twenty-first and 28 days CON, PAN+DYYG group and PAN+TAC group compared with PAN group, The weight of rats increased gradually, the difference was statistically significant (all P0.05). There was no significant difference between group PAN+DYYG and PAN+TAC group (P0.05). Seventh days, PAN group, PAN+DYYG group, PAN+TAC group and CON group were significantly reduced in urine volume, proteinuria, blood Cr and BUN value increased significantly (P0.05). Fourteenth days, PAN group, group, group and In group CON, urine volume decreased significantly, urine protein was 24 hours, blood Cr and blood BUN were lower than seventh days, while group PAN+DYYG, PAN+TAC group and PAN group, 24 hours urine protein quantitative, blood Cr and blood BUN value decreased more significantly (all P0.05). Twenty-first days, twenty-eighth days, PAN group, PAN+ DYYG group, 24 hours urine protein quantitative, blood and blood concentration by As compared with the PAN group, the decrease in the group PAN+DYYG and the PAN+TAC group was more obvious, the 24 hour urine volume increased significantly, and there was no significant difference between the PAN+DYYG group and the PAN+TAC group (P0.05). The expression of the foot cell marker protein Podcin in the PAN group was significantly lower than that in the CON group, and the PAN+DYYG and PAN+TAC group of the podpodic protein Podcin table was significantly lower than that in the PAN+TAC group. It was significantly higher than the PAN group, but there was no significant difference (P0.05). The immunofluorescence confocal microscopy showed that the fluorescence intensity of the foot cell specific skeleton protein Synaptopodin in the PAN group was significantly lower than that in the CON group, and the Synaptopodin fluorescence intensity of the foot cell specific skeleton protein of the PAN+DYYG group was significantly higher than that in the PAN group. The DYYG group was compared to the CON group. The transmission electron microscope showed that seventh days of PAN injection, PAN nephropathy model group, foot process fusion, lysosome necrosis, renal interstitial edema, PAN nephropathy given DYYG, TAC treatment group, widening of foot process, swelling.PAN injection, PAN nephropathy model group, more serious lesions, extensive fusion of foot process, partial foot process disappearing, and DYYG, TAC treatment group, Compared with the seventh days, the morphology of the foot process improved, but there was no significant difference between the two groups for twenty-first days. The PAN nephropathy group and the DYYG, TAC treatment group, the swelling of the foot process and the fusion improved. Compared with the PAN nephropathy model group, the improvement of the TAC treatment group was more obvious and similar to that of the CON group, but there was no significant difference between the two groups for twenty-eighth days, and DYYG, and there was no significant difference between the two groups. The morphological structure of foot process in the TAC treatment group returned to normal, but the width of the foot process in the PAN nephropathy model group was still higher than that of the CON group. Through the observation of the ultrastructure of the podocyte process, compared with the PAN nephropathy model group, the degree of foot process fusion in the TAC group was significantly lower than that of the PAN nephropathy model group, DYYG and TAC could improve and reverse the morphologic changes of the podocyte process. The fourth part: Western B. The results of lot showed that the expression of PTEN protein in the PAN group was down, the expression of PI3K, P Akt, m TOR increased obviously, the expression of LC3 II and Beclin decreased, the expression of P62 protein was increased, and the expression of signal pathway protein and autophagy related protein was significantly different from that of CON group. Up regulation, PI3K, P Akt, m TOR expression down-regulation; LC3 II, Beclin expression up-regulated, P62 protein expression reduced, signal pathway and autophagy related protein expression levels compared with the PAN group, the difference was statistically significant (P0.05). P62 expression (P0.05), but there is no significant difference in the expression of PTEN (P0.05). Conclusion: This study confirmed that PAN can inhibit autophagy of podgo, induced foot cell injury.DYYG intervention can reduce the inhibition of autophagy induced by PAN, increase the autophagy of podocyte and improve foot cell injury, and its protective mechanism may be by up regulation of PTEN expression and inhibiting PI3K/Akt/. The m TOR signal pathway is activated.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692

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