大黄素对早期糖尿病肾病大鼠足细胞上皮间质转化的影响

Effect of emodin on the epithelial-mesenchymal transition of podocytes in early diabetic nephropathy rats

目的探讨大黄素对早期糖尿病肾病(DN)大鼠足细胞上皮间质转化(EMT)的影响。方法选取健康雄性SD大鼠40只,按数字表法随机分为对照组10只和实验组30只。对照组大鼠实验过程中采用普通饲料喂养。实验组30只大鼠采用高糖、高脂饮食喂养4周后,一次性腹腔注射链脲佐菌素(40 mg/kg)的方法,制备DN模型,取造模成功的大鼠随机分为DN组和大黄素治疗组(DN+大黄素组)。造模成功后,DN组继续高糖、高脂饮食;DN+大黄素组给予高糖、高脂饮食,联合大黄素(20 mg/kg)灌胃,每天1次,连续8周。各组大鼠在实验14周末观测以下指标:(1)收集24 h尿液,测定24 h尿蛋白量;(2)完成尿液收集后称体质量并记录;(3)处死后取腹主动脉血检测肾肌酐、尿素氮、血糖;(4)取左侧肾脏,苏木精-伊红(HE)染色、过碘酸希夫染色(PAS)观察肾脏病理改变,免疫组织化学染色观察Nephrin、Desmin蛋白的表达情况;(5)取右侧肾脏肾皮质部位组织,电镜下观察足细胞及基底膜变化情况。结果实验组大鼠30只,DN大鼠模型造模成功24只,实验过程中DN组及DN+大黄素组大鼠各死亡2只。(1)14周末对照组、DN组、DN+大黄素组大鼠体质量分别为(350.1±14.16)、(265.2±7.62)、(312.1±11.77)g,差异有统计学意义(F=136.50,P<0.001)。(2)对照组肾肌酐、尿素氮、血糖、24 h尿蛋白分别为(28.88±4.36)μmol/L、(8.76±0.50)mmol/L、(9.81±0.86)mmol/L、(3.60±0.79)mg;DN组分别为(92.30±7.30)μmol/L、(29.41±6.67)mmol/L、(25.10±4.10)mmol/L、(35.23±4.07)mg;DN+大黄素组分别为(52.10±5.80)μmol/L、(14.93±1.03)mmol/L、(16.51±1.14)mmol/L、(13.35±1.89)mg。与对照组比较,DN组、DN+大黄素组肾肌酐、尿素氮、血糖、24 h尿蛋白量均升高;与DN组比较,DN+大黄素组各项指标均降低:差异均有统计学意义(P值均<0.01)。(3)HE染色观察肾组织形态学:对照组肾小球及肾小管结构未见异常;DN组肾小球硬化,肾小囊狭窄,部分肾小管上皮细胞空泡变性;DN+大黄素组肾小球硬化及肾小囊狭窄均有改善。(4)PAS染色观察肾小球毛细血管袢、系膜基质变化情况:对照组肾小球毛细血管袢结构清晰可见,系膜基质无异常;DN组肾小球毛细血管袢结构模糊,系膜基质增多;DN+大黄素组肾小球毛细血管袢结构较模糊,系膜基质较多。(5)免疫组织化学检测Nephrin及Desmin蛋白表达情况:与对照组比较,DN组、DN+大黄素组Nephrin蛋白光密度值均降低,Desmin蛋白光密度值均增加;与DN组比较,DN+大黄素组Nephrin蛋白光密度值增加,Desmin蛋白光密度值降低,差异均有统计学意义(P值均<0.01)。(6)透射电镜观察足细胞和基底膜变化情况:对照组足细胞轻度水肿,足突均匀、等宽排列,未见融合,基底膜完整,厚薄均一,3层结构明显;DN组足细胞呈中度水肿,足突明显融合、变宽,基底膜厚薄均一,3层结构明显;DN+大黄素组足细胞呈轻度水肿,足突融合、变宽情况减轻,基底膜厚薄均一。结论大黄素能显著改善早期DN大鼠的体质量、肾脏病理结构及功能,其机制可能与抑制足细胞发生EMT、促进肾病蛋白Nephrin的表达和减少足细胞EMT骨架蛋白Desmin表达有关。

Objective To explore the effect of emodin on podocyte epithelial-mesenchymal transition(EMT)in early diabetic nephropathy(DN)rats.Methods Forty adult healthy male Sprague-Dawley(SD)rats were randomly divided into control(n=10)and experimental(n=30)groups.Rats in the control group were fed with an ordinary diet during the experiment whereas those in the experimental group were fed with a high-glucose and high-fat diet combined with one-time intraperitoneal injection of streptozotocin(40 mg/kg)for 4 weeks to prepare the DN model.Mold success rats were randomly divided into DN and emodin treatment(DN+emodin)groups.The DN group continued to receive the high-fat and high-sugar diet,while the DN+emodin group was given a high-fat and high-sugar diet treated with emodin(20 mg/kg)once a day for 8 weeks.Rats in each group observed the following indexes at the end of the experimental 14 weeks:(1)Collect 24-hour urine volume of rats.Urine protein concentration of rats in 24 hours was calculated using urine protein quantitative test kit.(2)Weigh and record body weight after urine collection.(3)Extract blood of abdominal aorta to detect renal serum creatinine(SCr),serum urea nitrogen(BUN),and blood glucose(BG).(4)Select the left kidney for hematoxylin and eosin(HE)and periodic acid-Schiff(PAS)staining.Immunohistochemistry was applied to observe the expression of Nephrin and Desmin proteins.(5)Collect the renal cortex of the right kidney and observe changes of podocytes and glomerular basement membrane(GBM)under an electron microscope.Results The experimental group was composed of 30 rats,and 24 rats in the DN group were successfully modeled.Two rats in DN and DN+emodin groups died during the experiment.(1)Body weight of rats at the end of the 14th week in control,DN,and DN+emodin groups was(350.1±14.16),(265.2±7.62)and(312.1±11.77)g,respectively,with statistically significant differences(F=136.50,P<0.001).(2)The respective renal SCr,BUN,BG,and 24-hour urine protein were(28.88±4.36)μmol/L,(8.76±0.50)mmol/L,(9.81±0.86)mmol/L,and(3.60±0.79)mg in the control group;(92.30±7.30)μmol/L,(29.41±6.67)mmol/L,(25.10±4.10)mmol/L,and(35.23±4.07)mg in the DN group;and(52.10±5.80)mmol/L,(14.93±1.03)μmol/L,(16.51±1.14)mmol/L,and(13.35±1.89)mg in the DN+emodin group.Compared with those of the control group,Scr,BUN,BG,and 24-hour urine protein levels of DN and DN+emodin groups increased.All indexes decreased with statistical significance(all P values<0.01)in the DN+emodin group compared with those in the DN group.(3)Renal histomorphology via HE staining showed that glomerular and renal tubules were normal in the NC group.Glomerulosclerosis,renal capsule stenosis,and vacuolar degeneration of some renal tubular epithelial cells were observed in the DN group.Glomerulosclerosis and renal vesicle stenosis were significantly improved in the DN+emodin group.(4)PAS staining was used to observe the changes of glomerular capillary loops and mesangial matrix:The structure of glomerular capillary loops was clear and the mesangial matrix was normal in the NC group.Glomerular capillary loops were blurred and the mesangial matrix increased in the DN group.Glomerular capillary loops were fuzzy and the mesangial matrix increased in the DN+emodin group.(5)Immunohistochemical detection of Nephrin and Desmin protein expression presented that the optical density of Nephrin protein in DN and DN+emodin groups decreased while that of the Desmin protein increased compared with the control group.Compared with that of the DN group,the optical density increased in the Nephrin protein but decreased in the Desmin protein in the DN+emodin group,with statistically significant differences(all P values<0.01).(6)Changes of podocyte and GBM were observed via transmission electron microscopy.Podocytes showed mild edema and foot processes(FPs)were uniformly and equi-width arranged without fusion,the GBM was intact with uniform thickness,and an evident three-layer structure was observed in the NC group.Podocytes demonstrated moderate edema,the FP was fused and widened,the GBM was uniform,and the three-layer structure was evident in the DN group.Podocytes exhibited mild edema,FP fusion and widening were relieved,and the GBM showed uniform thickness in the DN+emodin group.Conclusion Emodin can significantly improve the body weight,renal pathological structure,and function of early DN rats.The mechanism may be related to inhibiting podocyte EMT,promoting the expression of nephropathy protein Nephrin and decreasing the expression of podocyte EMT skeleton protein Desmin.