周脂素在糖尿病肾病大鼠肾组织中的表达
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摘要
目的观察大鼠糖尿病肾病(diabetic nephropathy,DN)模型的特点和尿LN对早期DN的诊断价值,探讨周脂素(perilipin,Plin)在DN大鼠肾脏中的表达情况。方法将14只SD雄性大鼠随机分为对照组(普通饲料)和糖尿病肾病模型组(高糖高脂饲料),对照组6只,模型组8只,饲养4周后模型组按照30 mg/kg剂量注射1%链脲佐菌素(STZ),检测血糖≥16. 7 mmol/L,糖尿病模型制作成功,继续喂养6周,检测24 h尿蛋白≥30 mg/kg,糖尿病肾病模型制作成功。考马斯亮蓝检测24 h尿蛋白、ELISA测尿层粘连蛋白,HE染色观察肾组织的病理变化,Real-time PCR及Western blot检测肾脏组织中perilipin表达情况。结果模型鼠24 h尿蛋白≥30 mg/kg,糖尿病肾病大鼠模型制作成功。和对照组大鼠相比,模型组的肾重/体重比明显增高(P<0. 05),尿量、尿层粘连蛋白于5周出现升高、24 h尿蛋白于6周时出现升高,且三项指标均随着时间不断增高。肾组织病理检查显示:肾小球肥大,基膜增生,微小血管瘤形成,肾小管管腔变形,上皮脱落、空泡样变,大量单核、淋巴等炎性细胞浸润,间质内胶原纤维增生。模型组大鼠肾组织Plin的mRNA及蛋白表达均明显的升高(P<0. 05)。结论尿层粘连蛋白比24 h尿蛋白升高得早,可作为早期糖尿病肾病的警示指标。Plin表达增高可能参与了糖尿病肾病肾病变过程,为进一步探讨糖尿病肾病的发病机制提供新的思路。
Objective To characterize the rat model of diabetic nephropathy( DN),to evaluate the diagnostic value of urinary laminin for early detection of DN,and to investigate the expression of perilipin in diabetic rat kidneys.Methods Fourteen male Sprague-Dawley rats were randomly divided into a control group( regular diet,six animals) and a diabetic nephropathy model group( high sugar and high fat diet,eight animals). After feeding for 4 weeks,the rats of the disease model group were injected with a dose of 30 mg/kg of 1% streptozotocin. Induction of diabetes was considered successful when blood sugar levels were ≥ 16. 7 mmol/L. Upon induction of diabetes,animals were fed for an additional 6 weeks. Induction of diabetic nephropathy was considered successful when the 24-hour urinary protein level was ≥ 30 mg/kg.Coomassie brilliant blue( CBB) was used to determine the 24-hour urine protein levels,ELISA was used to measure urine laminin,and hematoxylin and eosin( H&E) staining was performed to observe the pathological changes in kidney tissues.Perilipin( Plin) expression in kidney tissues was determined by real-time PCR and western blotting. Results The detection of 24-hour urinary protein levels ≥ 30 mg/kg confirmed the successful induction of the rat model of diabetic nephropathy. The kidney-to-body weight ratio of the disease model group was increased significantly,when compared with the control diet group( P < 0. 05). Urinary volume and laminin increased by the 5 th week,while 24-hour urine protein increased by the 6 th week. All the three indicators were increasing over time. Pathological examination of the renal tissues revealed glomerular hypertrophy,basal membrane hyperplasia,microhemangioma formation,tubular cavity deformation,epithelial shedding and vacuolization, inflammatory monocyte and lymphocyte infiltration, and interstitial collagen deposition. A significant increase in Plin expression at the mRNA and protein levels in the kidney tissues of diabetic nephropathy rats was also observed. Conclusions Urinary laminin is increased earlier than the 24-hour urine protein level,thus can be used as an early biomarker of diabetic nephropathy. Increased Plin expression may play a role in the pathogenesis of diabetic nephropathy, and this protein therefore warrants further investigation to acquire a better understanding the molecular mechanisms underlying this disease.
Objective To characterize the rat model of diabetic nephropathy( DN),to evaluate the diagnostic value of urinary laminin for early detection of DN,and to investigate the expression of perilipin in diabetic rat kidneys.Methods Fourteen male Sprague-Dawley rats were randomly divided into a control group( regular diet,six animals) and a diabetic nephropathy model group( high sugar and high fat diet,eight animals). After feeding for 4 weeks,the rats of the disease model group were injected with a dose of 30 mg/kg of 1% streptozotocin. Induction of diabetes was considered successful when blood sugar levels were ≥ 16. 7 mmol/L. Upon induction of diabetes,animals were fed for an additional 6 weeks. Induction of diabetic nephropathy was considered successful when the 24-hour urinary protein level was ≥ 30 mg/kg.Coomassie brilliant blue( CBB) was used to determine the 24-hour urine protein levels,ELISA was used to measure urine laminin,and hematoxylin and eosin( H&E) staining was performed to observe the pathological changes in kidney tissues.Perilipin( Plin) expression in kidney tissues was determined by real-time PCR and western blotting. Results The detection of 24-hour urinary protein levels ≥ 30 mg/kg confirmed the successful induction of the rat model of diabetic nephropathy. The kidney-to-body weight ratio of the disease model group was increased significantly,when compared with the control diet group( P < 0. 05). Urinary volume and laminin increased by the 5 th week,while 24-hour urine protein increased by the 6 th week. All the three indicators were increasing over time. Pathological examination of the renal tissues revealed glomerular hypertrophy,basal membrane hyperplasia,microhemangioma formation,tubular cavity deformation,epithelial shedding and vacuolization, inflammatory monocyte and lymphocyte infiltration, and interstitial collagen deposition. A significant increase in Plin expression at the mRNA and protein levels in the kidney tissues of diabetic nephropathy rats was also observed. Conclusions Urinary laminin is increased earlier than the 24-hour urine protein level,thus can be used as an early biomarker of diabetic nephropathy. Increased Plin expression may play a role in the pathogenesis of diabetic nephropathy, and this protein therefore warrants further investigation to acquire a better understanding the molecular mechanisms underlying this disease.
引文
[1]黄璟,郭翔廷,张萍.上海某社区535例2型糖尿病患者现况分析[J].上海预防医学,2018,10(30):878-881.Huang J,Guo XT,Zhang P. Status analysis on 535 cases of type 2diabetes in a community in Shanghai[J]. Shanghai J Prev Med,2018,10(30):878-881.
[2] Sharaf El Din UAA, Salem MM, Abdulazim DO. Diabetic nephropathy:Time to withhold development and progression-A review[J]. J Adv Res,2017,8(4):363-373.
[3]陈春宇,陈志强,方敬,等.化瘀通络中药对糖尿病肾病大鼠肾组织纤维连接蛋白和层粘连蛋白的影响[J].天然产物研究与开发,2015,12(27):2134-2137.Chen CY,Chen ZQ,Fang J,et al. Effect of Huayu Tongluo Traditional Chinese Medicine on kidney fibronectin and laminin expression of diabetic nephropathy rats[J]. Nat Prod Res Dev,2015,12(27):2134-2137.
[4] Itabe H,Yamaguchi T,Nimura S,et al. Perilipins:a diversity of intracellular lipid droplet proteins[J]. Lipids Health Dis,2017,16(1):83.
[5] Eleftheriadis T, Antoniadi G, Pissas G, et al. The renal endothelium in diabetic nephropathy[J]. Ren Fail,2013,4(35):592-599.
[6] Delanghe JR,Delanghe SE,De Buyzere ML,et al. Infrared spectroscopic imaging for interrogating the carbohydrate biochemistry of diabetic nephropathy progression[J]. Kidney Int,2016,90(1):225-226.
[7]高雪,安至超,何其英,等.高脂饲料喂养时间对2型糖尿病肾病大鼠模型的影响[J].中国实验动物学报,2018,1(26):114-119.Gao X,An ZC,He QY,et al. Effects of high-fat diet feeding time on the establishment of a rat model of type 2 diabetic nephropathy[J]. Acta Lab Ani Sci Sin,2018,1(26):114-119.
[8] Kishore L,Singh R. Ameliorative effect of Cephalandra indica homeopathic preparation in STZ induced diabetic nephropathy rats[J]. J Ayurveda Integr Med,2018. pii:S0975-9476(17)30088-8.
[9] Masmiquel LL,Burgos R,Mateo C,et al. Effect of panretinal photocoagulation on serum levels of laminin in patients with diabetes:a prospective study[J]. Br J Ophthalmol,1999,83(9):1056-1059.
[10] Mason RR,Watt MJ. Unraveling the roles of Plin5:linking cell biology to physiology[J]. Trends Endocrinol Metab,2015,26(3):144-152.
[11]李凤娟,魏苏宁,王绿娅,等.雌激素抑制脂滴包被蛋白Perilipin 2减少肝细胞脂质沉积[J].心肺血管病杂志,2018,37(07):687-691.Li FJ, Wei SN, Wang LY, et al. Estrogen reduces lipid deposition in liver cells by inhibiting Perilipin 2[J]. J Cardiovasc Pulmon Dis,2018,37(07):687-691.
[12]樊林花,刘茂林,卫兵艳,等.周脂素和ADRP在糖代谢异常大鼠肝脏组织中的表达[J].中国病理生理杂志,2015,31(03):534-538.Fan LH,Liu ML,Wei BY,et al. Expression of perilipin and ADRP in rat liver tissues with abnormal glucose metabolism[J].Chin J Pathophys,2015,31(3):534-538.
[13]杜劲,侯娟妮,李秀川,等.脂滴包被蛋白5对高糖高脂诱导的小鼠心脏微血管内皮细胞凋亡的影响及机制[J].解放军医学杂志,2017,42(12):1045-1050.Du J,Hou JN,Li XC,et al. Effect of perilipin-5 on apoptosis of cardiac microvascular endothelial cells induced by high fat and high glucose in mice[J]. Med J Chin PLA,2017,42(12):1045-1050.
[2] Sharaf El Din UAA, Salem MM, Abdulazim DO. Diabetic nephropathy:Time to withhold development and progression-A review[J]. J Adv Res,2017,8(4):363-373.
[3]陈春宇,陈志强,方敬,等.化瘀通络中药对糖尿病肾病大鼠肾组织纤维连接蛋白和层粘连蛋白的影响[J].天然产物研究与开发,2015,12(27):2134-2137.Chen CY,Chen ZQ,Fang J,et al. Effect of Huayu Tongluo Traditional Chinese Medicine on kidney fibronectin and laminin expression of diabetic nephropathy rats[J]. Nat Prod Res Dev,2015,12(27):2134-2137.
[4] Itabe H,Yamaguchi T,Nimura S,et al. Perilipins:a diversity of intracellular lipid droplet proteins[J]. Lipids Health Dis,2017,16(1):83.
[5] Eleftheriadis T, Antoniadi G, Pissas G, et al. The renal endothelium in diabetic nephropathy[J]. Ren Fail,2013,4(35):592-599.
[6] Delanghe JR,Delanghe SE,De Buyzere ML,et al. Infrared spectroscopic imaging for interrogating the carbohydrate biochemistry of diabetic nephropathy progression[J]. Kidney Int,2016,90(1):225-226.
[7]高雪,安至超,何其英,等.高脂饲料喂养时间对2型糖尿病肾病大鼠模型的影响[J].中国实验动物学报,2018,1(26):114-119.Gao X,An ZC,He QY,et al. Effects of high-fat diet feeding time on the establishment of a rat model of type 2 diabetic nephropathy[J]. Acta Lab Ani Sci Sin,2018,1(26):114-119.
[8] Kishore L,Singh R. Ameliorative effect of Cephalandra indica homeopathic preparation in STZ induced diabetic nephropathy rats[J]. J Ayurveda Integr Med,2018. pii:S0975-9476(17)30088-8.
[9] Masmiquel LL,Burgos R,Mateo C,et al. Effect of panretinal photocoagulation on serum levels of laminin in patients with diabetes:a prospective study[J]. Br J Ophthalmol,1999,83(9):1056-1059.
[10] Mason RR,Watt MJ. Unraveling the roles of Plin5:linking cell biology to physiology[J]. Trends Endocrinol Metab,2015,26(3):144-152.
[11]李凤娟,魏苏宁,王绿娅,等.雌激素抑制脂滴包被蛋白Perilipin 2减少肝细胞脂质沉积[J].心肺血管病杂志,2018,37(07):687-691.Li FJ, Wei SN, Wang LY, et al. Estrogen reduces lipid deposition in liver cells by inhibiting Perilipin 2[J]. J Cardiovasc Pulmon Dis,2018,37(07):687-691.
[12]樊林花,刘茂林,卫兵艳,等.周脂素和ADRP在糖代谢异常大鼠肝脏组织中的表达[J].中国病理生理杂志,2015,31(03):534-538.Fan LH,Liu ML,Wei BY,et al. Expression of perilipin and ADRP in rat liver tissues with abnormal glucose metabolism[J].Chin J Pathophys,2015,31(3):534-538.
[13]杜劲,侯娟妮,李秀川,等.脂滴包被蛋白5对高糖高脂诱导的小鼠心脏微血管内皮细胞凋亡的影响及机制[J].解放军医学杂志,2017,42(12):1045-1050.Du J,Hou JN,Li XC,et al. Effect of perilipin-5 on apoptosis of cardiac microvascular endothelial cells induced by high fat and high glucose in mice[J]. Med J Chin PLA,2017,42(12):1045-1050.
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