抵当陷胸汤调控PI3K/Akt信号通路对糖尿病大鼠足细胞凋亡的影响

Influence of Didang Xianxiong Decoction on Apoptosis of Podocytes in Diabetes Rats Through PI3K/Akt Signaling Pathway

目的:观察抵当陷胸汤对糖尿病大鼠肾脏的保护作用及其对磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路的调控作用与对足细胞凋亡的影响,探讨抵当陷胸汤改善糖尿病肾病的机制。方法:采用单次腹腔注射链脲佐菌素(STZ)溶液55 mg·kg^(-1)的方法建立糖尿病模型,将模型复制成功的30只随机分为模型组、抵当陷胸汤组(8.10 g·kg^(-1))、小陷胸汤组(4.05 g·kg^(-1))、抵当汤组(4.05 g·kg^(-1))、阿拉氯胺(ALT-711)组(3 mg·kg^(-1)),每组6只,另设6只空白组大鼠。连续给药8周后,采用苏木素-伊红(HE)染色观察大鼠肾脏组织病理形态变化;马松(Masson)染色观察胶原沉积程度;高碘酸-席夫(PAS)染色观察基底膜病变;免疫组化法检测大鼠肾组织磷酸化(p)-PI3K、p-Akt蛋白表达;原位末端标记法(TUNEL)检测大鼠足细胞凋亡;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠肾组织PI3K、Akt mRNA表达情况;蛋白免疫印迹法(Western blot)检测肾组织中PI3K、p-PI3K、Akt、p-Akt、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷酸化糖原合成酶激酶-3β(p-GSK-3β)、胱天蛋白酶-3(Caspase-3)的蛋白表达。结果:与正常组比较,模型组大鼠肾小球代偿性膨胀,系膜细胞增殖,系膜间质大量胶原沉积,基底膜增厚,PI3K、Akt mRNA表达减少,PI3K、Akt蛋白磷酸化显著受到抑制(P<0.01),足细胞凋亡率显著升高(P<0.01),抗凋亡蛋白Bcl-2表达显著降低(P<0.01),Bax、p-GSK-3β、Caspase-3表达显著升高(P<0.01);与模型组比较,抵当陷胸汤组肾脏组织病理明显改善,PI3K、Akt mRNA表达显著升高(P<0.01),PI3K、Akt蛋白磷酸化水平显著升高(P<0.01),Bcl-2表达显著升高(P<0.01),Bax、p-GSK-3β、Caspase-3表达显著降低(P<0.01),足细胞凋亡率显著降低(P<0.01)。结论:抵当陷胸汤可能通过促进PI3K/Akt信号通路活化,抑制足细胞凋亡,减缓糖尿病肾病的发展进程。

Objective::To observe the protective effect of Didang Xianxiong decoction on the kidneys of diabetic rats,its regulation on the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway,and its influence on podocyte apoptosis and explore the mechanism of Didang Xianxiong decoction in improving diabetic nephropathy.M ethod::The diabetic model was established by a single intraperitoneal injection of streptozotocin(STZ)solution of 55 mg·kg^(−1).The successfully replicated model rats were randomly divided into the model group,Didang Xianxiong decoction group(8.10 g·kg^(−1)),Xiao Xianxiongtang group(4.05 g·kg^(−1)),Didangtang group(4.05 g·kg^(−1)),and alagebrium(ALT-711)group(3 mg·kg^(−1)),with six rats in each group.In addition,six rats were included in the blank group.After continuous administration for eight weeks,hematoxylin-eosin(HE)staining was used to observe the pathological changes in rats'kidney tissue.Masson staining was used to observe the degree of collagen deposition.Periodic acid-Schiff(PAS)staining was used to observe basement membrane lesions,and immunohistochemistry was used to detect the expression of phosphorylation(p)-PI3K and p-Akt proteins in rats'kidney tissue.The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)method was used to detect podocyte apoptosis.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of PI3K and Akt in rats'kidney tissue.Western blot was used to detect the protein expression of PI3K,p-PI3K,Akt,p-Akt,B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),phosphorylation glycogen synthase kinase-3β(p-GSK-3β),and Caspase-3 in the kidney tissue.Result::Compared with the normal group,the model group had compensatory expansion of glomeruli,proliferation of mesangial cells,a large amount of collagen deposition in the mesangial stroma,thickening of the basement membrane,decreased mRNA expression of PI3K and Akt,and inhibition of PI3K and Akt protein phosphorylation(P<0.01).It also underwent enhanced apoptotic signaling,decreased expression of anti-apoptotic protein Bcl-2(P<0.01),and increased expression of Bax,p-GSK-3β,and Caspase-3(P<0.01).Compared with the model group,Didang Xianxiong decoction significantly improved kidney tissue pathology,increased mRNA expression of PI3K and Akt(P<0.01),significantly up-regulated phosphorylation levels of PI3K and Akt proteins(P<0.01)and Bcl-2expression(P<0.01),downregulated the expression of Bax,p-GSK-3β,and Caspase-3(P<0.01),and weakened podocyte apoptotic signaling.Conclusion::Didang Xianxiong decoction may promote the activation of the PI3K/Akt signaling pathway,inhibit podocyte apoptosis,and thus slow down the progression of diabetic nephropathy.