阿托伐他汀调控PERK/eIF2α/CHOP通路减轻造影剂诱导的大鼠肾小管上皮细胞凋亡

目的研究造影剂肾病大鼠肾脏中葡萄糖调节蛋白78(GRP78)、内质网调节激酶(PERK)、真核起始因子2α(eIF2α)及C/EBP同源蛋白质(CHOP)的表达情况,探讨内质网应激在造影剂肾病发病中的作用及阿托伐他汀的干预作用。 方法60只大鼠随机分为4组：对照组、模型组和高、低剂量阿托伐他汀组(80 mg,40 mg),每组15只。分别于注射造影剂后24、48、72 h留取血清;检测各组大鼠的血清尿素氮(BUN)、血清肌酐(Scr);TUNEL法及Western印迹法测casepase-3的表达检测肾小管上皮细胞凋亡;免疫组化和Western印迹法检测各组大鼠肾组织GRP78、p-eIF2α、p-PERK及CHOP的表达。 结果与对照组相比,模型组大鼠BUN、Scr显著升高,细胞凋亡严重,GRP78、p-eIF2α、p-PERK及CHOP的表达均显著升高(P< 0.05);与模型组相比,高、低剂量阿托伐他汀组,BUN、Scr显著下降,凋亡指数降低,GRP78、p-eIF2α、p-PERK及CHOP的表达显著下调,但仍高于对照组,差异均达到统计学意义(P<0.05);高、低剂量阿托伐他汀组之间上述各指标差异均不显著。 结论PERK/eIF2α/CHOP通路介导的内质网应激可能参与大鼠造影剂肾病的发生发展;阿托伐他汀在造影剂诱导的肾脏损伤中发挥保护作用,这可能与其调节PERK/eIF2α/CHOP通路,从而减轻内质网应激有关。     

丹参酮ⅡA对慢性肾衰竭大鼠肾组织内质网应激相关分子GRP78、CHOP和Caspase-12表达的影响

目的:探究丹参酮ⅡA对慢性肾衰竭大鼠肾功能的保护作用机制。方法:60只SD雄性大鼠,随机分成5组,分别为:A正常组,B假手术组,C模型组,D模型+丹参酮ⅡA低剂量组,E模型+丹参酮ⅡA高剂量组。4周、8周、12周检测各组肌酐、尿素氮水平。12周时Tunel法检测细胞凋亡,Western blot法检测GRP78、CHOP和Caspase-12表达。结果:(1)C组较A组4周、8周、12周时肌酐、尿素氮升高(P0.05);D组、E组分别与C组比较:8周、12周时较模型组肌酐、尿素氮水平下降(P0.05);E组较D组12周时肌酐、尿素氮水平下降(P0.05)。(2)C组凋亡细胞较A组、B组增多(P0.05)。D组、E组凋亡细胞较C组减少(P0.05),E组较D组凋亡细胞减少(P0.05)。(3)C组与A组相比,GRP78、CHOP和Caspase-12表达均明显升高(P0.05);D组、E组与C组相比:GRP78、CHOP和Caspase-12表达均下降(P0.05);E组与D组比较:GRP78、CHOP和Caspase-12表达进一步下降(P0.05)。结论:TanⅡA能够改善CRF大鼠肾功能,抑制细胞凋亡,其机制可能是通过调节内质网应激相关分子GRP78、CHOP和Caspase-12表达水平实现。     

大鼠急性脊髓损伤后内质网应激相关蛋白CHOP的表达及其意义

目的:探讨内质网应激相关蛋白CHOP在急性脊髓损伤中的表达及其意义.方法:48只健康SD大鼠随机分为实验组与对照组(每组24只),实验组采用Allen法制作急性脊髓损伤模型,对照组只切开椎板不损伤脊髓.分别十伤后3h、1d、3d、7d时(每个时间点6只)应用改良Tarlov评分法评价其神经功能;然后处死动物,取受损节段脊髓行HE染色及免疫组化染色,观察形态学变化和CHOP在脊髓组织中的分布特点;并应用TUNEL方法检测神经细胞凋亡情况.其量的评定借助于IPP(Image-Pro Plus 6.0)软件,结果分别用累积光密度(integrated optical density,IOD)及平均光密度(mean density)表示.结果:对照组大鼠均末见明显瘫痪,实验组出现不同程度的下肢功能障碍,对照组各时间点的Tadov评分均高于实验组(P<0.01).对照组各时间点未见明显坏死细胞和细胞凋亡,实验组出现不同程度的细胞坏死和凋亡.实验组CHOP表达各时间点均明显高于对照组(P<0.01),实验组内CHOP表达1d增加,3d达到高峰,7d减少.实验组内细胞凋亡1d时增加,3d达到高峰,7d减少,且3h<1d<3d>7d(P<0.01).CHOP表达与细胞凋亡在时间上具有一致性,相关系数为0.644.结论:急性脊髓损伤能够诱导CHOP表达,CHOP表达的增高可能与内质网应激诱导的神经细胞凋亡有关.     

内质网应激相关凋亡途径参与单侧输尿管梗阻大鼠肾间质纤维化

目的 探讨内质网应激（ERS）相关凋亡途径在单侧输尿管梗阻（UUO）大鼠肾间质纤维化发生、发展中的作用。 方法 健康雄性Wistar大鼠25只,按随机数字表法分为UUO模型组（n=18）和假手术组（n=7）,UUO模型组行左侧输尿管结扎术,假手术组仅分离输尿管不结扎,分别于术后3 d、7 d、14 d处死各组大鼠,行HE和Masson染色,观察肾脏病理变化;比色法测定肾组织羟脯氨酸（HYP）含量;免疫组化法检测α平滑肌肌动蛋白（α-SMA）;原位末端标记法（TUNEL）与DNA电泳观察肾小管间质细胞凋亡情况;RT-PCR法检测梗阻侧肾组织ERS相关分子葡萄糖调节蛋白78（GRP78）mRNA表达变化;Western印迹法分析凋亡相关蛋白半胱氨酸天门冬氨酸蛋白酶3（caspase-3）和GRP78的蛋白表达变化。 结果 与假手术组比较,UUO模型组肾脏病理改变加重,肾间质纤维化程度随梗阻时间延长逐渐加重,肾组织HYP含量显著升高（P < 0.05）,肾组织α-SMA也在肾小管间质细胞广泛表达,TUNEL染色及DNA琼脂糖凝胶电泳提示大量的肾小管间质细胞凋亡。UUO模型组GRP78 mRNA表达于术后3 d即发生显著上调,而蛋白表达在术后7 d开始出现显著变化,与假手术组差异均有统计学意义（均P < 0.01）;在此后观察期间内GRP78 mRNA和蛋白均持续高水平表达。模型组大鼠肾组织caspase-3的蛋白表达在UUO术后3 d即有显著上调（P < 0.05）,且随着梗阻时间延长进行性升高,于术后7 d、14 d增多更为显著,与假手术组差异均有统计学意义（P < 0.05）。相关分析显示GRP78蛋白表达与肾组织HYP含量和caspase-3蛋白表达均呈正相关（r = 0.657,P < 0.01;r = 0.714,P < 0.01）。 结论 UUO早期即可诱导ERS标志蛋白表达变化,触发ERS。长期ERS可诱导肾小管间质细胞凋亡;caspase-3介导的ERS相关凋亡途径可能参与了肾间质纤维化过程。     

Klotho抑制内质网应激反应减轻UUO大鼠肾间质纤维化的实验研究

目的:观察可溶性Klotho蛋白对UUO模型大鼠肾脏内质网应激的影响。方法:24只清洁级SD大鼠随机分为假手术组、模型组、治疗组。模型组大鼠采用单侧输尿管梗阻(unilateral ureteral obstruction,UUO)方法建立肾间质纤维化模型;治疗组在UUO的基础上,给予可溶性KL蛋白腹腔注射,对照组及模型组给予等剂量生理盐水腹腔注射。14 d后检测各组大鼠的肾功能、肾脏病理改变;Western Blot检测肾组织Klotho、GRP78、CHOP、caspase-3的表达情况。结果:模型组大鼠肾功能恶化,肾脏病理损害明显,肾组织KL蛋白的表达明显下降,内质网相关蛋GRP78及凋亡相关蛋白CHOP、caspase-3的表达明显上升;治疗组大鼠肾功能好转,肾脏病理损害缓解,同时内质网应激相关蛋白及凋亡相关蛋白的表达明显下降。结论:KL可通过抑制ERS发挥肾脏保护作用。     

番茄红素对缺氧/复氧诱导小鼠心肌细胞内质网应激和凋亡的影响

目的 评价番茄红素对缺氧/复氧(hypoxia/reoxygenation,H/R)引起的小鼠心肌细胞内质网应激(endoplasmic reticulum stress,ERS)和凋亡的影响. 方法 建立C57BU6乳鼠心肌细胞H/R模型,采用随机数字表法将C57BL/6小鼠心肌细胞分为正常对照组(C组)、番茄红素组(Lyc组,含5μmol/L番茄红素的培养基预处理4h)、H/R组(H/R组,缺氧4h复氧6h)和番茄红素+H/R组(Lyc+H/R组,给予5μmol/L番茄红素预处理4h后行H/R处理).采用水溶性四氮唑-8(cell counting Kit-8,CCK-8)法检测心肌细胞存活率,倒置显微镜下观察细胞搏动频率,TUNEL法检测细胞凋亡率,二氯荧光素法(dichlomnuorescein diacetate,DCFH-DA)检测细胞内活性氧(reactive oxygen species,ROS)含量,实时荧光定量PCR (real-time PCR)检测葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、C/EBP同源蛋白(C/EBP homologous protein,CHOP)及半胱天冬氨酸蛋白酶-12 (cysteme aspartate specific protease-12,caspase-12)mRNA的表达,Western blot法分析剪切的半胱天冬氨酸蛋白酶-12(cleaved cysteme aspartate specific protease-12,Cleaved-caspase-12)和剪切的半胱天冬氨酸蛋白酶-3(cleaved cysteme aspartate specific protease-3,Cleaved-caspase-3)的表达. 结果 与C组比较,H/R组心肌细胞存活率显著降低[(100±5)％,(69±6)％](P＜0.01),搏动频率显著降低[(94±6),(28±5)次/min](P＜0.01),心肌细胞凋亡率[(4.9±1.5)％,(25.6±2.6)％]和ROS含量[(100±11)％,(226±10)％]显著升高(P＜0.01);CHOP mRNA[(1.00±0.10)、(2.60±0.19)]和GRP78 mRNA[(1.00±).18)、(4.12±0.23)]表达水平显著升高(P＜0.05);caspase-12 mRNA[(1.00±0.09)、(1.79±0.14)]、Cleaved-caspase-12[(1.00±0.08)、(1.85±0.10)]和Cleaved-caspase-3[(1.00±0.07)、(1.89±0.14)]表达水平显著升高(P＜0.05).与H/R组比较,Lyc+H/R组心肌细胞存活率明显升高[(69±6)％、(84±7)％](P＜0.05),搏动频率增加[(28±5)、(73±6)次/min] (P＜0.05),凋亡率显著降低[(25.6±2.6)％,(18.2±2.2)％](P＜0.05),细胞内ROS含量[(226±10)％、(140±16)％]明显降低,CHOP mRNA [(2.60±0.19)、(1.71±0.14)]和GRP78 mRNA[(4.12±0.23),(1.98±0.19)]表达水平显著降低(P＜O.05);caspase-12 mRNA[(1.79±0.14)、(1.38±0.11)]、Cleaved-caspase-12[(1.85±0.10)、(1.26±0.12)]和Cleaved-caspase-3[(1.89±0.14)、(1.36±0.12)]表达水平显著降低(P＜0.05). 结论 番茄红素可抑制H/R过程中的ERS及其凋亡信号途径而减轻心肌细胞损伤.     

祛痰通络汤对糖尿病大鼠肾组织内质网应激相关分子GRP78和CHOP表达的影响

目的:观察祛痰通络汤对糖尿病大鼠肾组织中内质网应激相关分子GRP78和CHOP表达的影响。方法:高糖高脂饲料联合腹腔注射STZ制备糖尿病模型,随机分为4组,分别为正常对照组、模型组、祛痰通络汤组、四苯基丁酸组,治疗8周。观察各组24 h尿蛋白定量、肾脏病理学变化;免疫组化和western-blot检测肾组织内质网应激相关蛋白GRP78,CHOP的表达。结果:祛痰通络汤降低尿蛋白,减轻病理改变;模型组肾组织的GRP78、CHOP表达明显升高(P0.05),祛痰通络汤组GRP78、CHOP表达明显低于模型组(P0.05)。结论:糖尿病大鼠肾脏中GRP78、CHOP表达明显增强,祛痰通络汤降低内质网应激,可能是其肾脏保护作用的重要机制。     

牛磺熊去氧胆酸对大鼠肝脏缺血再灌注损伤的保护作用

目的：探讨牛磺熊去氧胆酸（TUDCA）抗大鼠肝脏缺血再灌注（HIRI）损伤的作用及机制。 方法：将20只雄性SD大鼠随机均分为假手术组、TUDCA组、HIRI组、TUDCA+HIRI组,分别行假手术、TUDCA+假手术、HIRI造模,TUDCA+HIRI造模;TUDCA（250 mg/kg）于术前1 h灌胃给予,HIRI造模采用Pringle法（缺血60 min,再灌注12 h）。再灌注12 h后处死各组大鼠取材,观察肝组织病理学改变,检测血清谷丙转氨酶（ALT）水平,TUNEL法检测肝细胞凋亡,Western blot技术检测肝组织内质网应激分子糖调节蛋白78（GRP78）、p-真核细胞翻译起始因子2α（p-eIF2α）和C-EBP同源蛋白（CHOP）的表达。 结果：除假手术组与TUDCA组外,HIRI组与TUDCA+HIRI组大鼠肝组织均出现明显肝损伤病理改变,但TUDCA+HIRI组的损伤程度明显轻于HIRI组。与假手术组比较,HIRI组与TUDCA+HIRI组大鼠血清ALT水平明显升高,肝细胞凋亡和内质网应激分子GRP78、p-eIF2a和CHOP蛋白水平均明显升高（均P<0.05）,但TUDCA+HIRI组各项指标升高幅度均明显低于HIRI组（均P<0.05）;TUDCA组各项指标未见改变（均P>0.05）。 结论：TUDCA有抗大鼠肝HIRI的作用,其机制可能与抑制内质网应激反应有关。     

对比剂肾病大鼠肾脏GRP78与caspase-12表达及胰激肽原酶干预研究

目的:研究对比剂肾病大鼠肾脏中葡萄糖调节蛋白(glucose-regulated protein 78,GRP78)及半胱氨酸天冬氨酸蛋白酶12(caspase-12)的表达情况,探讨内质网应激在对比剂肾病发生发展中的作用,及胰激肽原酶的干预效果。方法:将84只大鼠随机分为3组:假手术组(D组),模型组(Z组),胰激肽原酶干预组(Y组)。自造模前3 d至处死日当天,Y组每天给予胰激肽原酶(15 U/kg)灌胃,D组及Z组每天给予等量生理盐水灌胃。分别于造模后12 h,24 h,48 h,72 h处死部分大鼠,观察各组大鼠血清尿素氮、肌酐水平变化;HE染色观察肾脏病理改变;原位末端转移酶标记法(TUNEL)检测肾脏细胞凋亡情况;RT-PCR检测肾脏GRP78 mRNA及caspase-12 mRNA的表达情况;免疫组化和Western-blot观察各组大鼠肾脏GRP78及caspase-12蛋白表达情况。结果:D组GRP78 mRNA、caspase-12 mRNA及GRP78、caspase-12几乎无表达,且肾组织无明显病理损伤及细胞凋亡;Z组病理变化明显,细胞凋亡较重,且以上指标均明显升高;Y组病理变化、细胞凋亡及GRP78 mRNA、caspase-12 mRNA、GRP78、caspase-12升高均较Z组明显减轻(P0.05),但仍高于D组(P0.05)。结论:内质网应激参与大鼠对比剂肾病的发生发展,胰激肽原酶可能通过减少caspase-12表达减轻内质网应激,发挥肾脏保护作用。     

内质网应激分子伴侣GRP78在大鼠缺血再灌注损伤肝脏中的表达

目的:观察内质网应激相关分子葡萄糖调节蛋白78(GRP78)在大鼠缺血再灌注损伤肝脏组织中的表达水平.方法:将24只健康雄性SD大鼠随机均分为假手术组,单纯肝缺血组(肝缺血30 min+再灌注0h),再灌注6h组(肝缺血30 min+再灌注6h)和再灌注12h组(肝缺血30 min+再灌注12h).分别检测各组血清丙氨酸转氨酶(ALT)和门冬氨酸转氨酶(AST)水平;肝组织病理学、凋亡情况及GRP78 mRNA表达水平.结果:与对照组比较,各实验组大鼠肝缺血后出现明显的肝组织损伤,且随着再灌注时间的延长损伤加重,表现为血清ALT和AST水平升高,明显的肝组织病理学改变,肝细胞凋亡率增加,各组间计量指标的差异均有统计学意义(均P＜0.05).大鼠肝组织GRP78 mRNA变化趋势与上述指标一致,缺血后表达明显上调,且随着再灌注时间延长而逐渐升高,各组间差异均有统计学意义(均P＜0.05).结论:缺血再灌注损伤肝脏组织中GRP78表达上调,但其具体作用还有待于探明.     

The protective effect of hydroxytyrosol on contrast - induced nephropathy and endoplasmic reticulum stress

Objective To investigate the expression of glucose-regulated protein 78(GRP78) and cysteine aspartic acid protease 12(Caspase - 12) and evaluate the endoplasmic reticulum stress (ERS) in rats with contrast - induced nephropathy (CIN), and observe the protective effects of hydroxytyrosol on CIN rats. Methods Eighty-four Wistar rats, (220±20) g, were randomly divided into control group, CIN group, hydroxytyrosol treated group (group C+H). At 12th, 24th, 48th, 72th day after the rats model were established, BUN and Scr were detected. ELISA were used to detect the expression of methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). HE staining were used to evaluate the pathological change of kidney. TUNEL were used to detect the apoptosis of tubular cells. Real-time PCR were used to detect the expression of GRP78 mRNA and Caspase-12 mRNA in tubular cells. Immunohistochemistry and Western blotting were used to detect the expression of GRP78 and Caspase - 12 protein in tubular cells. Results BUN, Scr, the mRNA and protein expression of GRP78, Caspase-12 in hydroxytyrosol treated group were higher than that in control group(P＜0.05), but were significantly lower than that in CIN group (P＜0.05). Pathological changes and the apoptosis of tubular cells in CIN group were more serious than that in hydroxytyrosol treated group (P＜0.05). Conclusions Endoplasmic reticulum stress may be associated with contrast-induced nephropathy. Hydroxytyrosol can protect kidney from contrast medium via reducing the endoplasmic reticulum stress.     

Klotho alleviates toxic effect of indoxyl sulfate on vascular endothelial cells and its mechanism

Objective To investigate the effect of klotho on the human vein umbilical endothelial cells (HUVECs) injury induced by indoxyl sulfate (IS) and to explore its mechanism and the role of endoplasmic reticulum stress (ERS) in this process. Methods (1) The cell vitalities of HUVECs incubated with different concentration of IS (5, 25, 50 mg/L) for 48 h and with 50 mg/L IS for different time points (12, 24, 48 h) were measured by CCK-8 assay. (2) HUVECs were incubated with 50 mg/L IS and different concentration of klotho (0, 1, 10, 100 μg/L) for 48 h and their cell viabilities were measured by CCK-8 assay. (3) HUVECs were divided into four groups: control group, IS group (50 mg/L IS), klotho group (50 mg/L IS+100 μg/L klotho) and Compound C group (50 mg/L IS+100 μg/L klotho+10 μmol/L Compound C). The cell vitality and the apoptosis of HUVECs were evaluated by CCK-8 assay and flow cytometry, respectively. The mRNA and protein expressions of GRP78 and CHOP were measured by real-time PCR and Western blotting. The phosphorylation level of AMPK was tested by Western blotting. Results IS inhibited cell vitality in the time-dependent and concentration-dependent manner. The cell viability of HUVECs with 50 mg/L IS was lower than normal control (P＜0.05). The inhibited cell vitality induced by IS was partly restored by klotho in concentration-dependent manner. The cell viability was higher in 100 μg/L klotho+50 mg/L IS group than 50 mg/L IS group (P＜0.05). Compared with control group, the expressions of GRP78 and CHOP and cell apoptosis increased, however, the level of phosphorylated AMPK (p-AMPK) decreased in IS group (all P＜0.05). Compared with IS group, the expressions of GRP78 and CHOP and cell apoptosis decreased and the level of p-AMPK increased in klotho group (all P＜0.05). Furthermore, the above effects of klotho could be partly blocked by Compound C. The above indexes showed statistical differences between Compound C group and klotho group. Conclusions IS can inhibit the HUVECs cell vitality, and induce ERS and cell apoptosis. Klotho protein could antagonize the above effects, probably through activating AMPK pathway and reducing ERS-mediated cell apoptosis.     

Relationship of angiotensin II type 1 receptor autoantibody and renal cell apoptosis induced by caspase-12 in diabetic nephropathy rats

Objective To study the relationship of angiotensin II type 1 receptor (AT1R) autoantibody (AT1-AA) and renal cell apoptosis induced by caspase-12 in diabetic nephropathy (DN) rats. Methods High-sucrose and high-fat diet and intraperitoneal injection of streptozotocin (35 mg/kg) were utilized to establish DN rat model. Serum AT1-AA was detected by enzyme-linked immunosorbent assay (ELISA) and renal cell apoptosis was detected by TUNEL staining. Furthermore, the mRNA levels of the endoplasmic reticulum stress (ERS) chaperone protein glucose regulated protein 78 (GRP78) and ERS-associated apoptosis protein caspase-12 were measured by real-time quantitative PCR. Additionally, the levels of GRP78 and caspase-12 protein were measured by Western blotting. Results The renal cell apoptosis rate in DN group was increased significantly (P＜0.01), and the renal cells apoptosis rate in AT1-AA positive DN group was higher than that in AT1-AA negative DN group [(20.05±1.71)% vs (13.24±4.93)%, P＜0.01]. The mRNA expressions of GRP78 and caspase-12 in DN group, in comparison to NC group, were increased significantly (P＜0.01), as well as the proteins (P＜0.01). And the expression of these mRNA and proteins had significant increment in AT1-AA positive DN rats when compared with AT1-AA negative DN rats (P＜0.05). Conclusions AT1-AA can induce ERS in the renal tissue of DN rats, and promote renal cell apoptosis likely via the modulation of caspase-12 signaling pathway.     

Lisinopril suppresses the endoplasmic reticulum stress associated tubular cell apoptosis induced by proteinuria in adriamycin nephropathy

Objective To investigate the role of endoplasmic reticulum stress in tubular epithelial cell apoptosis in chronic proteinuria rat model and the effect of lisinopril intervention. Methods Adriamycin nephropathy was induced in male Wistar rats (n=12) by a single injection of adriamycin at 2 mg/kg body weight. Rats were then randomly assigned to model group or treatment group, to which distilled water or lisinopril were administered respectively for 12 weeks. Six normal rats serving as controls were administered distilled water. 24 h urine samples were collected at week 4, 8, 12 and the urine protein was measured. At the end of study, serum was obtained and physiological parameters (serum creatinine, urea, total protein and albumin) were measured. Renal tubular epithelial cell apoptosis was assessed by TUNEL. GRP78, CHOP protein expression in kidney was quantified by immunohistochemistry and Western blotting. Results Compared to control group rats, increased proteinuria was observed in model group rats at week 4, 8, 12 (P＜0.05). Lisinopril treatment attenuated urine protein excretion significantly (P＜0.05). At week 12, hypoalbuminemia was detected in model group rats (P＜0.05), whereas the condition was alleviated by lisinopril (P＜0.05). There were no significant differences of serum creatinine, urea and total protein in each group (P＞0.05). Compared to control group rats, increased TUNEL positive tubular epithelial cells and tubular GRP78 and CHOP expression were also observed in model group rats (P＜0.05); however, these conditions in the kidney were significantly decreased in treatment group (P＜0.05). Conclusions Endoplasmic reticulum stress may be involved in the process of tubular epithelial cell apoptosis induced by proteinuria. Lisinopril may attenuate tubular epithelial cell apoptosis through regulating this signal pathway.     

低剂量电离辐射诱导小鼠睾丸细胞内质网应激及PERK-CHOP信号通路的激活

目的:探讨低剂量电离辐射与小鼠睾丸细胞内质网应激的发生以及PERK-CHOP通路激活的相关性。方法:健康雄性昆明小鼠随机分成时程-效应(75 mGy照射后0、3、6、12和24 h)和剂量-效应(0、50、75、100和200 mGy照射后12 h)组,每组动物10只。采用H2O2和MDA试剂盒比色法检测其含量;利用实时定量逆转录PCR(quantitative RT-PCR)检测GRP78、PERK和CHOP mRNA;Western印迹和图像分析技术检测GRP78、PERK、磷酸化PERK(phosphorylated PERK,pho-PERK)和CHOP蛋白表达。结果:小鼠经75 mGy全身照射后,睾丸组织中H2O2含量随时间延长而增加,MDA含量在3和6 h稍有降低,而后随时间延长而增加,二者在12和24 h较0 h时显著增加(P<0.05,P<0.01);除了GRP78 mRNA(3和24 h)和蛋白表达(6 h)分别在照射后有降低趋势外,GRP78(12 h)、PERK(3、6、12和24 h)和CHOP(12和24 h)的mRNA表达较0 h显著增加(P<0.05,P<0.01),GRP78(12和24 h)、pho-PERK(3、12和24 h)和CHOP(3、6、12和24 h)的蛋白表达也都较0 h显著增加(P<0.05,P<0.01),PERK蛋白表达则无明显变化规律。小鼠经50～200 mGy全身照射后12 h,睾丸组织中H2O2含量在50～100 mGy照射后随剂量增加而增加,200 mGy照射后则稍有降低,MDA含量随剂量增加而增加,而且H2O2含量(75和100 mGy)和MDA含量(75、100和200 mGy)显著高于0 mGy组(P<0.05,P<0.01);除了GRP78mRNA表达在50和200 mGy照射后有降低趋势外,GRP78(75和100 mGy)、PERK(75、100和200 mGy)和CHOP(50、75、100和200 mGy)的mRNA表达都显著高于0 mGy组(P<0.05,P<0.01),GRP78(100和200 mGy)、pho-PERK(50、100和200 mGy)和CHOP(50、75、100和200 mGy)的蛋白表达也都显著高于0 mGy组(P<0.05,P<0.01),而PERK蛋白表达则无明显变化规律。结论:低剂量电离辐射能够诱导小鼠睾丸细胞发生内质网应激,并且激活PERK-CHOP信号通路。     

内质网应激在白蛋白诱导肾小管上皮细胞凋亡中的作用

目的 探讨内质网应激在白蛋白超负荷诱导肾小管上皮细胞凋亡中的作用和分子机制。 方法 Western印迹法检测肾小管上皮细胞（HKC）内质网伴侣蛋白糖调节蛋白78（GRP78）和内质网应激蛋白CHOP（CCAAT增强子结合蛋白同源蛋白,也称为GADD153）表达与人血清白蛋白（HSA）作用时间和浓度的关系。实时荧光定量PCR法检测CHOP siRNA对CHOP基因转录的抑制情况。Western印迹法检测CHOP siRNA转染后CHOP蛋白水平的变化。膜联蛋白V和碘化丙锭（Annexin V-FITC和PI）双染的流式细胞术检测白蛋白诱导HKC凋亡的变化以及CHOP siRNA对HKC凋亡的影响。 结果 （1）分别以0、5、10、20 g/L白蛋白作用于HKC 24 h,GRP78、CHOP蛋白表达及细胞凋亡均随白蛋白浓度的增加而上调,各组间差异有统计学意义（P ＜ 0.01）;以20 g/L白蛋白分别作用于HKC 0、6、12、24、36 h,GRP78蛋白表达在6 h即显著增加,CHOP蛋白表达及HKC凋亡则在12 h显著增加,各组间差异有统计学意义（P ＜ 0.01）。（2）CHOP siRNA显著抑制白蛋白诱导的CHOP 基因转录及蛋白表达（P ＜ 0.01）,对白蛋白诱导的HKC凋亡有显著抑制作用（P ＜ 0.01）。 结论 白蛋白超负荷可诱导肾小管上皮细胞发生内质网应激,并通过上调促凋亡因子CHOP表达引起肾小管上皮细胞内质网相关的细胞凋亡,这可能是蛋白尿引起肾小管间质病变的重要机制。     

肾小管上皮细胞内质网应激时NGAL表达改变的调控机制研究

目的:探讨人肾小管上皮细胞(HK-2细胞)发生内质网应激时,NGAL表达增加的上游调控机制。方法:将HK-2细胞分为对照组(正常HK-2细胞),TG(毒胡萝卜素,thapsigargin)组(5μmol/L TG处理8 h),单纯转染组(siRNAATF4试剂转染24 h),转染+TG组(siRNA-ATF4试剂转染24 h后,5μmol/L TG处理8 h),阴性对照组(siRNA-阴性对照物转染24 h),DMSO组(5μmol/L DMSO处理8 h)。采用Western blot检测各组细胞内质网源性转录因子(CHOP)、内质网分子伴侣葡萄糖调节蛋白78(GRP78)、中性粒细胞明胶酶相关性载脂蛋白(NGAL)、激活转录因子4(ATF4)的表达,采用Real-time PCR方法测得ATF4mRNA、NGALmRNA表达量。结果:与对照组相比,TG组细胞NGAL、ATF4、ATF4mRNA、NGALmRNA表达量显著提高(P <0. 05),而转染+TG组、单纯转染组、阴性对照组、DMSO组中ATF4及NGAL差异无统计学意义(P> 0. 05)。与TG组相比,转染+TG组ATF4、NGAL、ATF4mRNA及NGALmRNA表达量呈显著降低趋势(P <0. 05)。在TG组与转染+TG组细胞中,CHOP和GRP78呈过表达状态(P <0. 05),而转染+TG组细胞CHOP和GRP78提升趋势明显低于TG组细胞(P <0. 05)。结论:(1) TG可诱导人肾小管上皮HK-2细胞发生内质网应激反应。(2) HK-2细胞发生内质网应激反应时,抑制ATF4表达会引起NGAL降低,提示ATF4是NGAL表达的上游调控因子。(3) HK-2细胞发生内质网应激反应时,抑制ATF4不能阻止CHOP和GRP78发生过表达,但可降低其升高程度,提示ATF4及NGAL降低可能对内质网应激反应介导HK-2细胞损伤起到一定的缓解作用。