摘要
目的 研究苦杏仁苷对糖尿病肾病(DKD)大鼠肾小管上皮细胞损伤及腺苷酸活化蛋白激酶/哺乳动物西罗莫司靶蛋白(AMPK/mTOR)通路的影响.方法 将SD大鼠分为空白组、模型组、苦杏仁苷低剂量组(3 mg·kg-1)、苦杏仁苷高剂量组(10 mg·kg-1)、Compound C 组(2.5 mg·kg-1 AMPK 抑制药Compound C)、苦杏仁苷高剂量+Compound C组(10 mg·kg-1苦杏仁苷+2.5 mg·kg-1 Compound C),每组10只.除空白组外,其余各组大鼠用连续喂养高脂高糖饲料+腹腔注射60 mg·kg-1 1％链脲佐菌素(STZ)方法进行DKD造模,造模成功后第2天给予相应药物处理,每天1次,持续给予8周,空白组、模型组均给予等体积0.9％NaCl.用二喹啉甲酸(BCA)法检测大鼠24 h尿蛋白含量,用血糖仪检测大鼠空腹血糖,用全自动生化分析仪检测大鼠血肌酸酐和血尿素氮水平,用原位末端标记法(TUNEL)染色检测大鼠肾组织细胞凋亡情况,用酶联免疫吸附测定(ELISA)法检测大鼠血清中肿瘤坏死因子-α(TNF-α)、白细胞介素1 β(IL-1 β)水平,用蛋白质印迹(Western Blot)法测定大鼠肾组织AMPK/mTOR通路、自噬及凋亡蛋白的表达水平.结果 空白组、模型组、苦杏仁苷低剂量组、苦杏仁苷高剂量组、Compound C组和苦杏仁苷高剂量+Compound C组的肾小管损伤评分分别为(0.43±0.05)、(3.26±0.24)、(1.92±0.14)、(0.73±0.06)、(3.81±0.29)和(1.78±0.12)分,细胞凋亡率分别为(5.23±0.41)％、(26.35±2.17)％、(16.76±1.42)％、(6.13±0.53)％、(35.68±3.08)％和(15.92±1.15)％,磷酸化 mTOR(p-mTOR)/mTOR 分别为0.31±0.03、0.80±0.08、0.60±0.06、0.38±0.04、0.92±0.08 和 0.69±0.07,磷酸化 AMPK(p-AMPK)/AMPK 分别为 0.91±0.08、0.39±0.04、0.56±0.06、0.88±0.07、0.23±0.02和0.52±0.05.模型组的上述指标与空白组、苦杏仁苷低剂量组、苦杏仁苷高剂量组相比,在统计学上差异均有统计学意义(均P＜0.05);苦杏仁苷高剂量组的上述指标与苦杏仁苷高剂量+Compound C组相比,在统计学上差异均有统计学意义(均P＜0.05).结论 苦杏仁苷可通过激活AMPK/mTOR信号通路,从而减轻DKD大鼠肾小管上皮细胞损伤.
Abstract
Objective To study the effect of amygdalin on renal tubular epithelial cell injury and adenosine monophosphate activated protein kinase/mammalian target of rapamycin(AMPK/mTOR)pathway in diabetic kidney disease(DKD)rats.Methods SD rats were grouped into blank group,model group,low dose amygdalin group(3 mg·kg-1),high dose amygdalin group(10 mg·kg-1),Compound C group(AMPK inhibitor Compound C 2.5 mg·kg-1),and high dose amygdalin+Compound C group(10 mg·kg-1 amygdalin+2.5 mg·kg-1Compound C),with 10 rats in each group.Except for blank group,the rats in other groups were fed with high-fat and high-glucose diet continuously+intraperitoneal injection of 60 mg·kg-1 1％streptozotocin(STZ)to construct the rat model of DKD.After the success of the model,the rats were given corresponding drug treatment the second day,once a day for 8 weeks,and the blank group and the model group were given equal volume physiological saline.Bicinchoninic acid(BCA)method was applied to detect 24-hour urine protein content in rats;glucose meter was applied to detect fasting blood glucose of rats;automatic biochemical analyzer was applied to detect the serum creatinine level and blood urea nitrogen level of rats;terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)staining was applied to detect apoptosis in kidney tissue cells;enzyme linked immunosorbent assay(ELISA)was applied to detect the levels of tumor necrosis factor-α(TNF-α)and interleukin-1 β(IL-1β)in serum;and Western Blot was used to measure the AMPK/mTOR pathway,autophagy,and apopt otic protein expression levels in kidney tissue.Results The renal tubule injury scores in blank group,model group,low dose amygdalin group,high dose amygdalin group,Compound C group and amygdalin high dose+Compound C group were 0.43±0.05,3.26±0.24,1.92±0.14,0.73±0.06,3.81±0.29 and 1.78±0.12,respectively;the apoptosis rates were(5.23±0.41)％,(26.35±2.17)％,(16.76±1.42)％,(6.13±0.53)％,(35.68±3.08)％and(15.92±1.15)％,respectively;phosphorylated mTOR(p-mTOR)/mTOR were 0.31±0.03,0.80±0.08,0.60±0.06,0.38±0.04,0.92±0.08 and 0.69±0.07,respectively;phosphorylated AMPK(p-AMPK)/AMPK were 0.91±0.08,0.39±0.04,0.56±0.06,0.88±0.07,0.23±0.02 and 0.52±0.05,respectively.There were statistically significant differences between model group and blank group,low dose amygdalin group,high dose amygdalin group(all P＜0.05).There were statistically significant differences between high dose amygdalin group and high dose amygdalin+Compound C group(all P＜0.05).Conclusion Amygdalin alleviates damage to renal tubular epithelial cells in DKD rats by activating the AMPK/mTOR signaling pathway.
基金项目
河北省沧州市重点研发指导基金(192106021)
国家科技期刊平台
NSTL
万方数据