三七调节肾脏PI3K/Akt/mTORC1-线粒体能量代谢治疗血瘀证慢性肾衰竭大鼠的机制研究

Mechanism of Notoginseng Radix et Rhizoma in regulating PI3K/Akt/mTORC1-mitochondrial energy metabolism to treat chronic renal failure in rats with blood stasis syndrome

观察三七对血瘀证阿霉素肾纤维化大鼠磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白复合物1(mammalian target of rapamycin complex 1,mTORC1)信号通路及线粒体能量代谢的影响,探讨三七发挥肾保护的作用机制。将30只造模成功的阿霉素肾纤维化雄性大鼠随机分为模型组、三七低剂量组、三七中剂量组、三七高剂量组、阳性对照组(盐酸贝那普利组),每组6只;将6只清洁级SD雄性大鼠分为正常组。正常组和模型组给予生理盐水,治疗组分别给予相应的药物,给药8周后,检测各组大鼠肾功能,肾脏病理,肾组织腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)含量和Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶活性,肾组织中ATP5B、mTORC1、p70核糖体S6激酶(70 kDa ribosomal protein S6 kinase,P70S6K)及P85、Akt、p-Akt、SH2域肌醇磷酸酶(SH2-containing inositol phosphatase,SHIP2)蛋白表达。结果显示,与正常组相比,模型组大鼠尿素氮(urea nitrogen,BUN)、血清肌酐(serum creatinine,SCr)水平均显著升高(P<0.01);与模型组相比,三七各剂量组和阳性对照组大鼠BUN、SCr水平均呈下降趋势,其中,三七中、高剂量组与阳性对照组差异具有统计学意义(P<0.05)。与模型组相比,三七各剂量组与阳性对照组肾组织的空泡样、纤维样改变,肾小球萎缩,肾小管囊性扩张,大量炎性细胞的浸润等病理改变均有不同程度的改善。与正常组比较,模型组肾组织线粒体ATP含量、Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg2+-ATP酶活性显著降低(P<0.05);与模型组比较,三七中、高剂量组和阳性对照组肾组织线粒体ATP含量、Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶活性显著升高(P<0.05)。与模型组相比,三七中、高剂量组以及阳性对照组ATP5B和SHIP2表达增加,mTORC1、P70S6K、P85、Akt和p-Akt表达降低,差异有统计学意义(P<0.05或P<0.01或P<0.001)。综上,三七可能通过抑制PI3K/Akt/mTORC1-线粒体能量代谢通路的活化,发挥抗纤维化机制,进而实现肾保护作用。

This study observed the effects of Notoginseng Radix et Rhizoma on the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin complex 1(mTORC1)signaling pathway and mitochondrial energy metabolism in the rat model of adriamycin-induced renal fibrosis with blood stasis syndrome to explore the mechanism of Notoginseng Radix et Rhizoma in protecting the kidney.Thirty male rats with adriamycin-induced renal fibrosis were randomized into model,low-,medium-,and highdose Notoginseng Radix et Rhizoma,and positive control groups(n=6).Six clean SD male rats were selected into the normal group.The normal group and model group were administrated with normal saline,and other groups with corresponding drugs.After 8 weeks of treatment,the renal function,renal pathology,adenosine triphosphate(ATP)levels,Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase activities,and the protein levels of ATP5B,mTORC1,70 kDa ribosomal protein S6 kinase(P70S6K),P85,Akt,p-Akt,and SH2-containing inositol phosphatase(SHIP2)in the renal tissue were determined.Compared with the normal group,the model group showed elevated levels of blood urea nitrogen(BUN)and serum creatinine(SCr)(P<0.01).Compared with the model group,Notoginseng Radix et Rhizoma and the positive control lowered the levels of BUN and SCr,which were significant in the medium-and high-dose Notoginseng Radix et Rhizoma groups and the positive control group(P<0.05).Compared with the model group,Notoginseng Radix et Rhizoma and the positive control alleviated the pathological changes in the renal tissue,such as vacuolar and fibroid changes,glomerulus atrophy,cystic expansion of renal tubules,and massive infiltration of inflammatory cells.Compared with the normal group,the model group showed decreased mitochondrial ATP content and Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase activities in the renal tissue(P<0.05),and medium-and high-dose Notoginseng Radix et Rhizoma and positive control mitigated such decreases(P<0.05).Compared with the model group,medium-and high-dose Notoginseng Radix et Rhizoma and the positive control up-regulated the protein levels of ATP5B and SHIP2 and down-regulated the protein levels of mTORC1,P70S6K,P85,Akt,and p-Akt(P<0.05 or P<0.01 or P<0.001).Notoginseng Radix et Rhizoma may exert an anti-fibrosis effect by inhibiting the activation of the PI3K/Akt/mTORC1 pathway to restore mitochondrial energy metabolism,thus protecting the kidney.