1.西南医科大学中西医结合学院;2.西南医科大学附属中医医院;3.乐山市中医医院
四川省科技计划项目；四川省中医药管理局项目
1.College of Integrated Traditional Chinese and Western Medicine,Southwest Medical University;2.Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University;3.Leshan Hospital of Traditional Chinese Medicine
Science and Technology Program of Sichuan Province; Sichuan Provincial Administration of Traditional Chinese Medicine Project
【摘要】目的 探讨芪石肾舒胶囊对早期糖尿病肾病小鼠肾纤维化的治疗作用和机制。 方法 采用多次注射链脲佐菌素建立糖尿病肾病小鼠模型。小鼠随机分为正常组、模型组和芪石组(0.9 g/kg/d)，每组8只，连续灌胃4周，监测每周空腹血糖。4周后，检测尿微量白蛋白肌酐比、血肌酐和血尿素氮含量；苏木精-伊红染色、过碘酸雪夫染色和天狼星红染色观察肾脏病理变化情况；实时荧光定量逆转录聚合酶链式反应检测纤维连接蛋白、Ⅰ型胶原蛋白α1及α-平滑肌肌动蛋白的信使核糖核酸(mRNA)表达水平；免疫组化和免疫蛋白印迹法检测纤维连接蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、α-平滑肌肌动蛋白、足突蛋白、肾病蛋白及转化生长因子-β1/SMAD家族成员2/3 (TGF-β1/Smad2/3)通路相关蛋白含量。 结果 与正常组相比，模型组小鼠空腹血糖和尿微量白蛋白肌酐比水平升高(P＜0.001)；肾组织出现系膜增生、基底膜增厚和胶原沉积；纤维连接蛋白、Ⅰ型胶原蛋白α1及α-平滑肌肌动蛋白的mRNA水平升高(P＜0.05)；足突蛋白和肾病蛋白水平下降(P＜0.05)，纤维连接蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、α-平滑肌肌动蛋白、足突蛋白、肾病蛋白及TGF-β1/Smad2/3通路蛋白水平升高(P＜0.05)；与模型组相比，芪石组尿微量白蛋白肌酐比水平降低(P＜0.05)，肾脏病理损伤有所缓解；纤维连接蛋白、Ⅰ型胶原蛋白α1及α-平滑肌肌动蛋白的mRNA水平下降(P＜0.05)，足突蛋白和肾病蛋白水平升高(P＜0.05)，纤维连接蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、α-平滑肌肌动蛋白及TGF-β1/Smad2/3通路蛋白水平均降低(P＜0.05)。 结论 芪石肾舒胶囊可改善糖尿病肾病小鼠肾纤维化，其机制可能与抑制TGF-β1/Smad2/3信号通路有关。
【Abstract】Objective To investigate the therapeutic effect and underlying mechanism of Qishishenshu capsule on renal fibrosis in mice with early diabetic nephropathy (DN). Methods DN mouse model was established by multiple injection of streptozotocin (STZ). The mice were randomly divided into the normal group (NC), model group (DN) and Qishi group (QS)(0.9 g/kg/d), 8 mice in each group, gavaged continuously for 4 weeks, and fasting blood glucose (FBG) was measured weekly. 4 weeks later, urinary microalbumin creatinine ratio (UACR), serum creatinine (SCr) and blood urea nitrogen (BUN) were measured. Hematoxylin-eosin staining (HE staining), periodic acid-Schiff staining (PAS staining) and Sirius red staining were used to analyze renal pathological changes. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the Message RNA (mRNA) levels of fibronectin (FN), collagen type I alpha 1 (Col1a1), and α-smooth muscle actin (α-SMA). Immunohistochemistry (IHC) and Western blot (WB) were performed to detect FN, collagen type I (Collagen I), collagen type III (Collagen III), α-SMA, Podocin, Nephrin and transforming growth factor-β1/SMAD family member2/3 (TGF-β1/Smad2/3) pathway-related proteins. Results Compared with the NC group, mice in the DN group showed significantly higher levels of FBG and UACR (P<0.001). Mesangial hyperplasia, basement membrane thickening and collagen deposition occurred in renal tissue. The mRNA levels of FN, Col1a1 and α-SMA were increased (P<0.05). Protein levels of Podocin and Nephrin were decreased (P<0.05). The levels of FN, Collagen I, Collagen III, α-SMA and TGF-β1/Smad2/3 pathway protein were increased (P<0.05). Compared with the DN group, the level of UACR in QS group was decreased (P<0.05), and renal pathological injury was alleviated. The mRNA levels of FN, Collagen1 and α-SMA were attenuated (P<0.05). The protein levels of Podocin and Nephrin were elevated (P<0.05). Protein levels of FN, Collagen I, Collagen III, α-SMA and TGF-β1/Smad2/3 pathway protein were also decreased (P<0.05). Conclusions Qishishenshu Capsules improved renal fibrosis in DN mice probably through the inhibition of TGF-β1/Smad2/3 signaling pathway.
