四味姜黄汤散对糖尿病肾病大鼠PI3K/Akt信号通路的调节作用

Regulating effect of Siwei-Jianghuangtang powder on PI3K/Akt signaling pathway in diabetic nephropathy rats

目的探究四味姜黄汤散对DN大鼠肾脏的保护作用,以及对磷脂酰肌醇3-激酶(phosphatidyl inositol 3-kinase, PI3K)/蛋白激酶B(protein kinase B, PKB/Akt)信号通路的影响。方法将大鼠按随机数字表法分为假手术组,模型组,四味姜黄汤散低、中、高剂量组(以下简称"低、中、高剂量组")及卡托普利组,每组12只。除假手术组外,其余各组大鼠制备糖尿病模型。造模结束后,四味姜黄汤散低、中、高剂量组大鼠灌胃四味姜黄汤散12、24、48 mg/g,卡托普利组灌胃卡托普利溶液30 mg/kg,假手术组及模型组灌胃等体积生理盐水,1次/d,连续给药8周。给药4周、8周测定大鼠尿液中24 h尿蛋白,采用酶联免疫法检测血清TG、TC、BUN、SCr水平,末次给药结束后取肾组织,测定肾脏肥大指数,HE、Masson染色观察大鼠肾组织病理学改变,电镜观察肾组织超微结构变化;Westerin blot法检测肾组织中胰岛素受体底物2(insulin receptor substrate 2, IRS-2)、p-PI3K、p-Akt蛋白表达情况;免疫组化法检测肾组织中肾病蛋白(Nephrin)、足突蛋白质(Podocin)蛋白阳性表达情况。结果与模型组比较,四味姜黄汤散低、中、高剂量组大鼠体重升高,肾脏肥大指数及第4、8周24 h尿蛋白降低(P<0.05),血清TC、TG、SCr、BUN降低(P<0.05),肾小球硬化评分、肾小管间质损伤评分、肾小管间质胶原纤维比例均降低(P<0.05);肾组织p-PI3K蛋白[(0.42±0.06)、(0.61±0.09)、(0.76±0.05)比(0.21±0.03)]、IRS-2蛋白[(0.34±0.03)、(0.61±0.10)、(0.78±0.09)比(0.24±0.03)]、p-Akt蛋白[(0.28±0.06)、(0.43±0.07)、(0.69±0.05)比(0.15±0.03)]表达升高(P<0.05);Nephrin阳性表达率[(31.78±3.14)%、(42.54±5.15)%、(50.97±7.13)%比(22.86±3.07)%]、Podocin阳性表达率[(33.67±4.59)%、(50.54±11.41)%、(61.03±7.55)%比(22.64±2.97)%]升高(P<0.05)。结论四味姜黄汤散可缓解DN大鼠肾脏损伤,其机制可能与激活PI3K/Akt信号通路,缓解足细胞损伤有关。

Objective To explore the protective effect of Siwei-Jianghuangtang powder on kidney of diabetic nephropathy rats and its influences on phosphatidyl inositol 3-kinase/protein kinase B signaling pathway (PI3K/Akt). Methods The SD rats were divided into 6 groups, sham operation group, model group, Siwei-Jianghuangtang powder low, middle and high dose groups (12, 24, 48 mg/kg Siwei-Jianghuangtang powder) and positive group (Captopril, 30mg/kg), with 12 rats in each group. The 24h urinary protein in urine of rats was measured after 4 weeks and 8 weeks of administration. The enzyme linked immunosorbent assay was used to detect serum lipid level and renal function. After the last administration, the renal tissue was obtained, and the renal hypertrophy index was measured. The HE and Masson staining were used to observe the histopathological changes of kidney in rats, and the ultrastructural changes of renal tissue were observed by electron microscope. The western blot (WB) was used to analyze the expressions of IRS-2, p-PI3K and p-Akt in renal tissues, and the immunohistochemistry was used to detect the positive expressions of nephrin and podocin protein in kidney. Results Compared with the model group, the p-PI3K protein (0.42 ± 0.06, 0.61 ± 0.09, 0.76 ± 0.05 vs. 0.21 ± 0.03), the IRS-2 protein (0.34 ± 0.03, 0.61 ± 0.10, 0.78 ± 0.09 vs. 0.24 ± 0.03) and the P-Akt protein (0.28 ± 0.06, 0.43 ± 0.07, 0.69 ± 0.05 vs. 0.15 ± 0.03) in Siwei-Jianghuangtang powder low, middle and high dose group significantly increased (P<0.05). The positive expression rate of Nephrin (31.78%± 3.14%, 42.54%± 5.15%, 50.97%± 7.13% vs. 22.86%± 3.07%) and the positive expression rate of Podocin (33.67%± 4.59%, 50.54%± 11.41%, 61.03%± 7.55% vs. 22.64%± 2.97%) significantly increased (P<0.05). Conclusions The Siwei-Jianghuangtang powder can relieve renal injury of diabetic nephropathy rats. Its mechanism may be related to activating PI3K/Akt signaling pathway and alleviating podocyte injury.