苗药地锦草对糖尿病肾病大鼠肾小球足细胞相关蛋白表达的影响

Effect of Euphorbia humifusa on expression of glomerular podocyte-related proteins in diabetic kidney disease rats

目的观察苗药地锦草对糖尿病肾病(diabetic kidney disease,DKD)大鼠肾小球足细胞相关蛋白Nephrin、Desmin和Angptl4表达的影响,明确其在DKD治疗中的作用机制。方法30只8周龄雄性SD大鼠随机分为:正常对照组(Normal,N组),DKD组(D组),DKD地锦草治疗组(Euphorbia humifusa,E组)。D组与E组大鼠单次尾静脉注射链脲佐菌素(Streptozobocin,STZ)(55 mg/kg)复制DKD模型,N组注射溶媒柠檬酸缓冲液,连续3次24 h尿白蛋白量>5 mg/d可确定为糖尿病肾病成模,5周后D组10只大鼠及E组9只大鼠DKD成模。DKD模型复制成功后,E组大鼠灌胃法服用地锦草300 mg/(kg·d),D组与N组灌胃给予同体积普通双蒸水,各组大鼠灌胃共16周。所有大鼠在处死前称重、每周收集大鼠留尿检测24 h尿白蛋白、留血检测血清白蛋白、尿素氮、肌酐、胆固醇、白蛋白;苏木素-伊红(HE)染色、马松三色染色(Masson)观察肾病理学结构;透射电镜观察足细胞的形态;WT1染色定位足细胞并采用disector/fractionator方法行足细胞计数;免疫组化观察Nephrin、Desmin、Angptl4的蛋白定位;Western blot法检测Nephrin、Desmin、Angptl4、Collagen IV的蛋白表达量;qPCR法检测Nephrin、Desmin、Angptl4、Collagen IV的mRNA含量。结果D组较N组大鼠血糖、24 h尿白蛋白、尿素、肌酐、胆固醇均明显升高,白蛋白明显降低(P<0.05)。E组大鼠上述各指标均较D组明显改善(P<0.05)。HE及Masson染色可见,N组大鼠肾小球结构完成,系膜细胞及基质正常。D组大鼠肾小球系膜细胞增生,系膜区基质沉积较多。E组大鼠经地锦草灌胃治疗后上述情况得以改善。透射电镜显示N组大鼠肾小球足细胞足突正常,D组大鼠足突融合或消失,E组大鼠足突较完整。WT1染色显示D组足细胞计数较N组减少,E组较D组明显增加(P<0.05)。免疫组化,Western blot及qPCR结果显示,Desmin、Angptl4及Collagen IV蛋白及mRNA在N组表达较少,上述指标在D组表达明显增多(P<0.05),E组大鼠经地锦草治疗后Desmin、Angptl4及Collagen IV蛋白及mRNA的表达较D组明显减少(P<0.05)。D组Nephrin蛋白及mRNA表达较N组明显减少,E组较D组表达增多(P<0.05)。结论地锦草治疗可以明显减少DKD大鼠足细胞Desmin、Angptl4及Collagen IV的表达并增加Nephrin的表达,有效保护足细胞的机械屏障及电荷屏障,抑制足细胞发生转分化,减轻DKD大鼠的蛋白尿并改善其肾功能。

Objective To observe the effect of Euphorbia humifusa on expression of nephrin,desmin,and Angptl4 in glomerular podocytes of diabetic kidney disease(DKD)rats,and to clarify the mechanism of its action in DKD treatment.Methods Eight-week-old male SD rats were randomly divided into normal(N),DKD group(D)and DKD treated with Euphorbia humifusa(E)groups.Rats in D and E groups were treated by a single tail vein injection of 55 mg/kg streptozotocin to induce the DKD model.Rats in the N group were injected with the same volume of citrate buffer.The DKD model was evaluated by 24 h urinary albumin of>5 mg/d for 3 consecutive detection.After 5 weeks,the DKD model was established in 10 D group rats and nine E group rats.After successfully establishing the DKD model,the E group was administered 300 mg/(kg·d)Euphorbia humifusa by gavage,and rats in D and N groups were administered the same volume of double distilled water by intragastric administration for 16 weeks.All rats were weighed before death,urine was collected to measure 24 h urinary albumin,and blood was collected for serum albumin,urea nitrogen,creatinine,cholesterol,and albumin measurements.Hematoxylin-eosin and Masson trichromatic staining were used to observe the nephrotic structure.Podocyte morphology was observed by transmission electron microscopy.Podocytes were labeled by WT1 staining and counted by the dissector/fractionator method.Protein localization of nephrin,desmin,Angptl4,and WT1 was observed by immunohistochemistry.Western blot was used to measure the protein expression levels of nephrin,desmin,Angptl4,and collagen IV.Nephrin,desmin,Angptl4,and collagen IV mRNA expression was measured by qPCR.Results Compared with the findings in the N group,in the D group,blood glucose,24-hour urine albumin,urea nitrogen,creatinine,and cholesterol were significantly increased,while albumin was significantly decreased.All indexes in the E group were significantly improved compared with those in the D group(P<0.05).Hematoxylin-eosin and Masson staining showed that the glomerular structure of rats in the N group was complete without obvious mesangial hyperplasia.In the D group,mesangial hyperplasia and mesangial matrix deposition were observed.In E the group,these conditions were improved by intragastric treatment with Euphorbia humifusa.Transmission electron microscopy showed that glomerular podocytes of the N group were normal,podocytes of the D group were fused or disappeared,and podocytes of the E group were intact.WT1 staining showed that the number of podocytes in the D group was lower than that in the N group,and the podocytes number in the E group was significantly higher than that in the D group(P<0.05).Immunohistochemistry,western blotting,and qPCR showed that protein and mRNA expression of desmin,Angptl4,and collagen IV in the N group were lower than those in the D group,while protein and mRNA expression of desmin and Angptl4 in the E group were significantly lower than those in the D group(P<0.05).Nephrin protein and mRNA expression in the D group was significantly lower than that in the N group,and nephrin protein and mRNA expression in the E group was higher than that in the D group(P<0.05).Conclusions Euphorbia humifusa treatment significantly reduces the expression of desmin,Angptl4,and collagen IV,and increases the expression of nephrin in DKD rat podocytes.Euphorbia humifusa effectively protected the mechanical and charge barrier of podocytes,inhibits transdifferentiation of podocytes,reduces proteinuria,and improves renal functions.