[关键词]
[摘要]
目的 观察白首乌C-21甾苷对单侧输尿管结扎所致的大鼠肾纤维化的干预作用,并探究其作用机制。方法 80只SD大鼠随机选取15只为假手术组,剩余大鼠进行单侧输尿管结扎制备大鼠肾纤维化模型,将造模大鼠随机分为4组,即模型组、阳性药氯沙坦钾(20 mg/kg)组及白首乌C-21甾苷高、低剂量(400、200 mg/kg)组,每组15只。各组大鼠于造模后第2天起,每天ig给药1次(10 mL/kg),连续给药4周,假手术组与模型组大鼠ig给予等体积蒸馏水。干预28 d后,每组取10只大鼠的肾组织,采用碱水解法检测肾组织中羟脯氨酸(Hyp)水平,采用HE和Masson染色检测病理损伤和纤维化程度,通过免疫荧光、Western blotting法检测转化生长因子-β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)和E-钙黏蛋白(E-cadherin)的表达,通过实时荧光定量PCR(qRT-PCR)法测定肾组织中I型胶原蛋白(collagen-I)和collagen-III mRNA表达水平。结果 与模型组比较,白首乌C-21甾苷能明显改善肾纤维化大鼠的病变程度,减少炎性细胞浸润、纤维组织和胶原的增生,可以明显降低大鼠肾脏指数,降低肾组织中Hyp水平,并呈剂量依赖性(P<0.05、0.01);免疫组化和Western blotting结果显示,白首乌C-21甾苷可以明显上调大鼠肾组织E-cadherin的表达水平,下调TGF-β1和α-SMA蛋白的表达水平(P<0.05、0.01);qRT-PCR结果显示白首乌C-21甾苷可显著降低大鼠肾组织collagen-I、collagen-III mRNA表达水平(P<0.01)。结论 白首乌C-21甾苷对单侧输尿管结扎的大鼠肾纤维化具有明显的干预作用,其机制可能与抑制collagen-I和collagen-III的过度表达,下调TGF-β1、α-SMA蛋白高表达和上调E-cadherin蛋白低表达有关,通过调控肾小管上皮-间质转化,发挥抗肾纤维化的作用。
[Key word]
[Abstract]
Objective To investigate the effects of C-21 steroidal glucosides from Cynanchum auriculatum on renal fibrosis in rats caused by unilateral ureteral ligation and explore its mechanisms. Methods A total of 15 rats were randomly chosen as sham operation group (Sham), while the remaining rats underwent unilateral ureteral ligation. The rats after the operation were randomly divided into four groups, namely the Sham operation group, the model group, the positive control group (20 mg/kg), C. auriculatum high group (400 mg/kg), C. auriculatum low group (200 mg/kg), 15 per group, and the rats in each group were administrated with corresponding drugs. After the intervention of 28 d, all the rats were sacrificed and the kidneys were then removed. The content of hydroxyproline was measured. HE and Masson staining were conducted to assess kidney pathological changes and renal fibrosis. The protein expression of TGF-β1, α-SMA, and E-cadherin were tested with immunohistochemistry and Western blotting. The mRNA expression of collagen-I and collagen-III was evaluated using qRT-PCR. Results Compared with the model group, C-21 steroid glycosides significantly alleviated the kidney pathological injury and renal fibrosis, and reduced fibrous tissue and collagen proliferation. C-21 steroid glucosides from C. auriculatum can significantly reduce the kidney/body weight ratio and the content of hydroxyproline in kidney tissue (P<0.05, 0.01), and it showed in a dose-dependent manner. Compared with the model group, the expression of α-SMA and TGF-β1 was decreased significantly, and the expression of E-cadherin was increased significantly (P<0.05, 0.01) in C. auriculatum group. Moreover, compared with the model group, C-21 steroidal glucosides significantly down-regulated the mRNA expression of collagen-I and collagen-III mRNA (P<0.01). Conclusion C-21 steroid glycosides from C. auriculatum can effectively attenuate renal fibrosis in rats with unilateral ureteral ligation and its underlying mechanisms may be related to inhibiting the over-expression of collagen-I and collagen-III, down-regulating the expression of α-SMA and TGF-β1, and up-regulating the expression of E-cadherin. By regulating the tubular epithelial-mesenchymal transition, it exerts its anti-fibrotic effect.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(81774178);国家自然科学基金资助项目(81373888);江苏省研究生创新项目(SJCX19-0369)