基于SIRT1/PGC-1α通路探讨葡萄籽原花青素提取物对糖尿病肾脏疾病大鼠的保护作用

Protective effects of grape seed proanthocyanidin extract on rats with diabetic kidney disease from the perspective of SIRT1/PGC-1αpathway

目的探讨葡萄籽原花青素提取物(GSPE)通过调控沉默信息调节因子1(SIRT1)/过氧化物酶体增殖物激活受体γ辅助活化因子1α(PGC-1α)通路对糖尿病肾脏疾病(DKD)大鼠的保护作用。方法将40只雄性SD大鼠随机分为对照组、对照治疗组、模型组和治疗组,每组10只。腹腔注射链脲佐菌素(STZ)制备糖尿病大鼠模型,对照治疗组和治疗组灌胃GSPE,对照组和模型组灌胃等量生理盐水,连续给药12周,检测大鼠血糖、血肌酐和尿微量白蛋白;过碘酸希夫(PAS)染色观察肾组织病理学变化;透射电镜观察肾组织超微结构;采用Tunel染色法评估肾组织细胞凋亡水平;采用免疫组化法和Western blotting法检测肾组织中线粒体生物合成SIRT1/PGC-1α通路相关分子SIRT1、PGC-1α、核呼吸因子1(NRF1)和线粒体转录因子A(TFAM)的表达水平。结果模型组血糖[(39.38±4.18)mmol/L vs(8.21±3.57)mmol/L]、血肌酐[(55.83±3.72)μmol/L vs(40.00±2.49)μmol/L]和尿微量白蛋白[(10.98±3.36)mg/L vs(1.22±0.23)mg/L]水平较对照组升高(P<0.05)。模型组肾组织损伤严重,线粒体碎片化增多,细胞凋亡[(31.81±8.84)%vs(0.50±0.35)%]多于对照组(P<0.05);同时肾脏中线粒体生物合成相关分子SIRT1(0.34±0.13 vs 0.66±0.06)、PGC-1α(0.32±0.03 vs 0.71±0.13)、NRF1(0.05±0.01 vs 0.21±0.02)和TFAM(0.06±0.03 vs 0.33±0.06)蛋白表达水平较对照组降低(P<0.05)。经GSPE干预后,治疗组血糖[(27.26±3.93)mmol/L vs(39.38±4.18)mmol/L]、血肌酐[(43.50±1.70)μmol/L vs(55.83±3.72)μmol/L]和尿微量白蛋白[(4.05±2.06)mg/L vs(10.98±3.36)mg/L]水平较模型组下降(P<0.05),细胞凋亡[(4.90±1.62)%vs(31.81±8.84)%]减少(P<0.05),肾脏中SIRT1(0.55±0.05 vs 0.34±0.13)、PGC-1α(0.62±0.14 vs 0.32±0.03)、NRF1(0.16±0.02 vs 0.05±0.01)和TFAM(0.26±0.06 vs 0.06±0.03)蛋白表达水平较模型组升高(P<0.05)。结论GSPE可能通过调控线粒体生物合成SIRT1/PGC-1α信号通路,改善DKD大鼠肾脏线粒体生物合成,从而发挥肾脏保护作用。

Objective To explore the protective effects of grape seed proanthocyanidin extract(GSPE)on rats with diabetic kidney disease(DKD)via silent information regulator 1(SIRT1)/peroxisome proliferator-activator receptorγcoactivator 1α(PGC-1α)signaling pathway.Methods A total of 40 male Sprague Dawley(SD)rats were randomly divided into control,control treatment,model and treatment groups,with 10 rats in each group.The diabetic rat models were induced by intraperitoneal injection of streptozotocin(STZ).Rats in the control treatment and treatment groups received GSPE gavage,while rats in the control and model groups were given equivalent amount of normal saline for 12 weeks.The blood glucose,serum creatinine and urinary microalbumin were detected.The pathological changes of renal tissue were observed with periodic acid Schiff(PAS)staining.Mitochondrial morphology was observed with a transmission electron microscopy.The apoptosis of renal tissue cells was evaluated with Tunel staining.The protein levels of SIRT1/PGC-1αsignaling pathway-related molecules of mitochondrial biogenesis,including SIRT1,PGC-1α,nuclear respiratory factor 1(NRF1),and mitochondrial transcription factor A(TFAM)were determined with immunohistochemistry and Western blotting,respectively.Results Compared with the control group,the model group had significantly increased blood glucose[(39.38±4.18)mmol/L vs(8.21±3.57)mmol/L],serum creatinine[(55.83±3.72)μmol/L vs(40.00±2.49)μmol/L]and urinary microalbumin[(10.98±3.36)mg/L vs(1.22±0.23)mg/L](P<0.05).Compared with the control group,the model group had severer histopathological damages and mitochondrial fragmentation in the renal tissue,more significant apoptosis[(31.81±8.84)%vs(0.50±0.35)%](P<0.05),but significantly lower expressions of SIRT1(0.34±0.13 vs 0.66±0.06),PGC-1α(0.32±0.03 vs 0.71±0.13),NRF1(0.05±0.01 vs 0.21±0.02),and TFAM(0.06±0.03 vs 0.33±0.06)(P<0.05).Compared with the model group,the treatment group had significantly decreased levels of blood glucose[(27.26±3.93)mmol/L vs(39.38±4.18)mmol/L],serum creatinine[(43.50±1.70)μmol/L vs(55.83±3.72)μmol/L],urinary microalbumin[(4.05±2.06)mg/L vs(10.98±3.36)mg/L]and cell apoptosis[(4.90±1.62)%vs(31.81±8.84)%](P<0.05),but significantly increased expressions of SIRT1(0.55±0.05 vs 0.34±0.13),PGC-1α(0.62±0.14 vs 0.32±0.03),NRF1(0.16±0.02 vs 0.05±0.01),and TFAM(0.26±0.06 vs 0.06±0.03)(P<0.05).Conclusion GSPE may improve mitochondrial biogenesis in DKD rats by regulating SIRT1/PGC-1αsignaling pathway,and thus playing a protective role.