知母皂苷元通过AMPK-mTOR-ULK1途径抑制肾小球系膜基质合成和激活自噬治疗糖尿病肾病的研究

目的探究知母皂苷元基于AMPK-mTOR-ULK1途径对糖尿病肾病大鼠的干预作用。方法将40只SD大鼠随机分为对照组、模型组、知母皂苷元低剂量组、知母皂苷元高剂量组和二甲双胍组,每组8只。除对照组外,其余组采用高脂饲料饲喂联合链脲佐菌素(STZ)诱导建立糖尿病模型。知母皂苷元低、高剂量组大鼠分别灌胃20 mg/(kg·d)和60 mg/(kg·d)知母皂苷元,二甲双胍组灌胃500 mg/(kg·d)二甲双胍,其余组灌胃等量生理盐水,连续8周。检测各组大鼠血糖、24 h尿蛋白、尿素氮、肌酐水平,HE染色和PAS染色观察大鼠肾脏病理组织学变化,检测肾组织中α-SMA表达情况和Ⅰ型胶原、Ⅳ型胶原、纤维连接蛋白含量,Western blot法检测肾组织中Beclin-1、LC3、AMPK、p-AMPK、mTOR、p-mTOR、ULK1和p-ULK1表达量。结果与对照组比较,模型组大鼠血糖、24 h尿蛋白、尿素氮、肌酐水平和肾重/体重均显著增高(P均<0.05);肾脏组织结构紊乱,肾小球基底膜增厚;肾小球体积、系膜基质面积、α-SMA阳性面积和Ⅰ型胶原、Ⅳ型胶原、纤维连接蛋白含量及p-mTOR/mTOR表达量均显著升高(P均<0.05),Beclin-1、LC3-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK、p-ULK1/ULK1表达量均显著降低(P均<0.05)。与模型组比较,二甲双胍组大鼠血糖和知母皂苷元低剂量组、知母皂苷元高剂量组、二甲双胍组大鼠24 h尿蛋白、尿素氮、肌酐水平和肾重/体重均显著降低(P均<0.05);肾脏病理组织学变化得到明显改善;肾小球体积、系膜基质面积、α-SMA阳性面积和Ⅰ型胶原、Ⅳ型胶原、纤维连接蛋白含量及p-mTOR/mTOR表达量均显著降低(P均<0.05),Beclin-1、LC3-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK、p-ULK1/ULK1表达量均显著增高(P均<0.05)。结论知母皂苷元可通过AMPK-mTOR-ULK1途径抑制肾小球系膜基质合成和激活自噬改善大鼠糖尿病肾病。

Sarsasapogenin inhibits glomerular mesangial matrix synthesis and activates autophagy to improve diabetic nephropathy through the AMPK-mTOR-ULK1 pathway

Objective It is to explore the intervention effect of sarsasapogenin on diabetic nephropathy based on the AMPK-mTOR-ULK1 pathway.Methods Forty SD rats were randomly divided into control group,model group,low and high-dose groups of sarsasapogenin,and metformin group,with 8 rats in each group.Except for the control group,the other groups were fed with high-fat feed combined with streptozotocin(STZ)induction to establish diabetic models.The rats in the low and high doses of sapogenin groups were given 20 mg/(kg·d)and 60 mg/(kg·d)sapogenin by gavage respectively,the metformin group was given 500 mg/(kg·d)metformin by gavage,the other groups were given the same amount of normal saline by gavage,all the groups were treated for 8 weeks.The levels of blood glucose,24h urine protein,urea nitrogen,creatinine were detected in each group,the histopathological changes of rat kidneys were observed by HE staining and PAS staining,and the expression of α-SMA and the content of type Ⅰ collagen,type Ⅳ collagen and fibronectin in kidney tissue were detected,the expression of Beclin-1,LC3,AMPK,p-AMPK,mTOR,p-mTOR,ULK1 and p-ULK1 in kidney tissue were detected by Western blot method.Results Compared with the control group,the levels of blood glucose,24 h urine protein,urea nitrogen,creatinine and kidney weight/body mass of the rats were significantly increased in the model group(all P<0.05);the kidney tissue structure was disordered,and the glomerular basement membrane was thickened;the globule volume,mesangial matrix area,α-SMA positive area,and the contents of type Ⅰ collagen,type Ⅳ collagen,fibronectin,and p-mTOR/mTOR expression were all significantly increased(all P<0.05),the expression levels of Beclin-1,LC3-Ⅱ/LC3-Ⅰ,p-AMPK/AMPK,and p-ULK1/ULK1 were significantly reduced(all P<0.05).Compared with the model group,the blood glucose of rats in the metformin group and the levels of 24 h urine protein,urea nitrogen,creatinine and kidney weight/body mass of rats in the anemarrhena low dose group,anemarrhena high dose group,and metformin group were significantly decreased(all P<0.05);kidney histopathological changes were significantly improved;glomerular volume,mesangial matrix area,α-SMA positive area,the cotents of type Ⅰ collagen,type Ⅳ collagen,fibronectin and p-mTOR/mTOR expression were significantly decreased(all P<0.05),the expression levels of Beclin-1,LC3-Ⅱ/LC3-Ⅰ,p-AMPK/AMPK,and p-ULK1/ULK1 were significantly increased(all P<0.05).Conclusion Sarsasapogenin couhd inhibit glomerular mesangial matrix synthesis and activates autophagy through the AMPK-mTOR-ULK1 pathway,thus to improve diabetic nephropathy in rats.