枸杞多糖对糖尿病肾病兔足细胞形态结构和nephrin蛋白的影响研究
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摘要
目的观察枸杞多糖(LBP)对糖尿病肾病(DN)兔足细胞形态结构和nephrin蛋白的表达的影响。方法雄性日本大耳白兔,随机分为正常组(4只)和DN模型组(16只),后者糖尿病(DM)造模成功后随机分为阴性组(4只)、预防组(4只)、治疗组(4只)、阳性组(4只),持续饲喂高脂饲料(1%胆固醇+4%猪油)2周,构建DN模型。正常组灌胃0.9%生理盐水1 ml/(kg·d)连续12周,阴性组DM模型成功后灌胃0.9%生理盐水1 ml/(kg·d)连续12周,预防组DM模型成功后灌胃LBP 10 mg/(kg·d)持续12周,阳性对照组DM模型成功后第8周开始灌胃美卡素3.7 mg/(kg·d)持续4周,治疗组DM模型成功后第8周开始灌胃LBP 10 mg/(kg·d)持续4周。在12周末处死动物取肾脏,做组织切片,电镜下观察。取肾脏测肾组织中的nephrin蛋白及mRNA表达。结果正常兔足细胞足突细密,栅栏状,紧贴在毛细血管基底膜外面。基底膜连续平滑,无增厚。足细胞无深染,细胞核无固缩无裂解。糖尿病DN兔足细胞肥大,染色深。细胞核可见裂解,核仁异染。足突大面积融合,基底膜(GBM)广泛裸露,足突增粗、倒伏、弯曲增多,基底膜中重度增厚,不规则增厚。LBP干预组DN兔足细胞肿胀程度减轻,细胞核浓集现象减轻,足突融合情况改善。与正常组相比,各组nephrin蛋白mRNA的表达明显下降,差异存在极显著性(P<0.01),与阴性组、治疗组、阳性组相比,预防组nephrin蛋白mRNA的表达较多,差异有极显著性(P<0.01)。结论 LBP可以明显改善DN兔足细胞的凋亡情况,减少足突的融合,基底膜的增厚,并且可以拮抗糖尿病所导致的nephrin蛋白表达的下降。
Objective To observe the effect of lycium barbarum polysaccharides( LBP) on themorphological structure of DN rabbit podocytes and expression of nephrin proteins. Methods Male Japanese big ear rabbits were divided into normal group( 4 rabbits) and diabetic nephropathy,( DN) model group( 16 rabbits). The model group was divided into negative group( 4 rabbits),prevention group( 4 rabbits),treatment group( 4 rabbits),positive group( 4 rabbits) after successful modeling. Except for the normal group,each group was rendered into DM mold,fed with high fat( 1% cholesterol + 4% lard) 2 w continuously to build DN model. Normal group was given 0. 9% saline 1 ml/( kg·d)continued until age 12 weeks,as well as negative DM model group. DM models of prevention group were given LBP 10 ml/( kg·d),continued until age 12 weeks,positive control group was given Micardis 3. 7 mg/( kg·d) at age 8 w and sustained 4 w after successfully built model. The treatment group was given LBP 10 mg/( kg·d) at age 8 w and sustained 4 w. The animals at the end of 12 w was executed. Their renal biopsy under confocal microscope was observed. The expression of nephrin protein and mRNA in renal tissue was measured. Results The podocytic process of normal rabbit were fine,palisade and close to the outside of the capillary basement membrane. Basement membrane were continuous and smooth,no thickening. Podocytes were not stained and nuclei without condensation and cracking. Podocytic processes of diabetes DN rabbits were hypertrophic and stained dark,there were visible cleavage nucleus and nucleolus were metachromatic. A large area of podocytic process fused,GBM extensively exposed. Podocytic processes were thicken,lodged and bending increased. The thickness of basement membrane were severely and irregularly thicken. Swelling of DN rabbit podocytes of LBP intervention group were relieved. The phenomenon of nuclear enrichment was reduced,podocytic processes fusion was improved. Changes in nephrin mRNA expression in each group compared with the control group,nephrin protein mRNA expression in each group significantly decreased,with significant differences( P < 0. 01). Compared with the negative group,treatment group,positive group,nephrin protein mRNA expression in prevention group is more than them,the difference was significant( P < 0. 01). Conclusion LBP can significantly improve podocyte apoptosis,reducing podocytic process fusion and thickening of the basement membrane,and can antagonize drop nephrin protein expression caused by diabetes.
Objective To observe the effect of lycium barbarum polysaccharides( LBP) on themorphological structure of DN rabbit podocytes and expression of nephrin proteins. Methods Male Japanese big ear rabbits were divided into normal group( 4 rabbits) and diabetic nephropathy,( DN) model group( 16 rabbits). The model group was divided into negative group( 4 rabbits),prevention group( 4 rabbits),treatment group( 4 rabbits),positive group( 4 rabbits) after successful modeling. Except for the normal group,each group was rendered into DM mold,fed with high fat( 1% cholesterol + 4% lard) 2 w continuously to build DN model. Normal group was given 0. 9% saline 1 ml/( kg·d)continued until age 12 weeks,as well as negative DM model group. DM models of prevention group were given LBP 10 ml/( kg·d),continued until age 12 weeks,positive control group was given Micardis 3. 7 mg/( kg·d) at age 8 w and sustained 4 w after successfully built model. The treatment group was given LBP 10 mg/( kg·d) at age 8 w and sustained 4 w. The animals at the end of 12 w was executed. Their renal biopsy under confocal microscope was observed. The expression of nephrin protein and mRNA in renal tissue was measured. Results The podocytic process of normal rabbit were fine,palisade and close to the outside of the capillary basement membrane. Basement membrane were continuous and smooth,no thickening. Podocytes were not stained and nuclei without condensation and cracking. Podocytic processes of diabetes DN rabbits were hypertrophic and stained dark,there were visible cleavage nucleus and nucleolus were metachromatic. A large area of podocytic process fused,GBM extensively exposed. Podocytic processes were thicken,lodged and bending increased. The thickness of basement membrane were severely and irregularly thicken. Swelling of DN rabbit podocytes of LBP intervention group were relieved. The phenomenon of nuclear enrichment was reduced,podocytic processes fusion was improved. Changes in nephrin mRNA expression in each group compared with the control group,nephrin protein mRNA expression in each group significantly decreased,with significant differences( P < 0. 01). Compared with the negative group,treatment group,positive group,nephrin protein mRNA expression in prevention group is more than them,the difference was significant( P < 0. 01). Conclusion LBP can significantly improve podocyte apoptosis,reducing podocytic process fusion and thickening of the basement membrane,and can antagonize drop nephrin protein expression caused by diabetes.
引文
[1]李菁菁,罗琼,闫俊,等.枸杞多糖对糖尿病肾病兔肾功能及MCP-1 ICAM-1mRNA表达的影响[J].营养学报,2015,60(1):32-36,40.
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[7]李朝晖,马晓鹂,吴万征.枸杞多糖降血糖作用的细胞实验研究[J].中药材,2012,35(1):124-127.
[8]黄云兰,李诺利,梁耿,等.枸杞多糖对链脲佐菌素联合高糖高脂饲养致2型糖尿病大鼠的影响[J].右江民族医学院学报,2012,34(6):745-746
[9]刘峰,黄国栋,朱凌燕,等.枸杞多糖对2型糖尿病大鼠胰腺β细胞PDX-1基因表达的实验研究[J].中国医药科学,2013,3(1):34-36,45.
[10]加杨娥,任立汆,燕华玲.枸杞多糖对糖尿病患者的疗效研究进展[J].世界最新医学信息文摘(连续型电子期刊),2016,16(52):54-55.
[11]丁园.枸杞多糖对2型糖尿病大鼠血糖、血脂的影响[J].辽宁医学院学报,2015,36(5):12-14.
[12]夏惠,唐华丽,潘佳琪,等.枸杞多糖防治2型糖尿病的机制研究进展[J].食品科学,2016,37(9):232-236.
[2]赵岩.枸杞多糖对糖尿病大鼠早期肾组织保护作用及机制初探[J].光明中医,2016,31(12):1729-1731.
[3]陈小明,黄丹丹,李自成.新西兰兔糖尿病肾病模型的建立方法[J].中国病理生理杂志,2012,28(4):765-768.
[4]于蕾,崔宏伟.枸杞多糖对糖尿病大鼠的降血糖作用及血清TNF-α、IL-6水平的影响[J].浙江临床医学,2015,17(12):2062-2064.
[5]李晓冰,陈玉龙,展俊平,等.枸杞多糖对糖尿病大鼠血糖水平及机体免疫功能的影响[J].中国全科医学,2013,16(27):3208-3210
[6]李长江,张英锋,高大威,等.枸杞多糖分离纯化及降血糖效果的研究[J].燕山大学学报,2014,52(6):557-560.
[7]李朝晖,马晓鹂,吴万征.枸杞多糖降血糖作用的细胞实验研究[J].中药材,2012,35(1):124-127.
[8]黄云兰,李诺利,梁耿,等.枸杞多糖对链脲佐菌素联合高糖高脂饲养致2型糖尿病大鼠的影响[J].右江民族医学院学报,2012,34(6):745-746
[9]刘峰,黄国栋,朱凌燕,等.枸杞多糖对2型糖尿病大鼠胰腺β细胞PDX-1基因表达的实验研究[J].中国医药科学,2013,3(1):34-36,45.
[10]加杨娥,任立汆,燕华玲.枸杞多糖对糖尿病患者的疗效研究进展[J].世界最新医学信息文摘(连续型电子期刊),2016,16(52):54-55.
[11]丁园.枸杞多糖对2型糖尿病大鼠血糖、血脂的影响[J].辽宁医学院学报,2015,36(5):12-14.
[12]夏惠,唐华丽,潘佳琪,等.枸杞多糖防治2型糖尿病的机制研究进展[J].食品科学,2016,37(9):232-236.