木犀草素含药血清对高糖诱导肾小球系膜细胞AMPK/SIRT-1/PGC-1α信号通路及凋亡的影响

Effects of Serum Containing Luteolin on AMPK/SIRT-1/PGC-1αSignal Pathway and Apoptosis of Mesangial Cells Induced by High Glucose

目的:探讨木犀草素(Luteolin)含药血清对高糖诱导下肾小球系膜细胞(GMCs)凋亡及单磷酸腺苷活化蛋白激酶(AMPK)/沉默信息调节因子1(SIRT1)/过氧化物酶增殖物激活受体γ共激活因子1α(PGC-1α)信号通路的影响。方法:将健康SPF级SD雄性大鼠分为正常组、Luteolin低(50 mg/kg)、中(100 mg/kg)、高(200 mg/kg)剂量组、罗格列酮阳性组(0.36 mg/kg),每组10只,均灌胃给予相应剂量药物,1次/d,连续7 d后,经腹主动脉取血制备含药血清。取SV40 MES13型小鼠GMCs将其分为正常组、高糖模型组、Luteolin含药血清低、中、高剂量组、阳性含药血清组,各组细胞按相应剂量药物处理并培养24 h后,采用四甲基偶氮唑蓝(MTT)法及流式细胞仪检测各组GMCs存活率及凋亡率;以蛋白免疫印迹法(Western Blot)检测各组细胞通路蛋白AMPK/SIRT-1/PGC-1α及凋亡相关蛋白脂肪酸合酶(Fas)、B淋巴细胞瘤-2基因(Bcl-2)表达。结果:与正常组相比,模型组GMCs细胞密度、胞质微丝样结构、胞核颗粒沉积等HE染色病理形态、存活率、Bcl-2蛋白表达均升高(P<0.05),凋亡率、pAMPK/AMPK、SIRT-1、PGC-1α、Fas蛋白表达均降低(P<0.05)。与模型组相比,Luteolin含药血清低、中、高剂量组及阳性对照组细胞密度、胞质微丝样结构、胞核颗粒沉积等HE染色病理形态、存活率、Bcl-2蛋白表达均降低(P<0.05),凋亡率、pAMPK/AMPK、SIRT-1、PGC-1α、Fas蛋白表达均升高(P<0.05),且Luteolin各剂量组呈剂量依赖性。阳性对照组与Luteolin含药血清高剂量组相比,上述指标无差异(P>0.05)。结论:Luteolin血清可能通过激活AMPK/SIRT-1/PGC-1α蛋白表达,抑制高糖诱导下GMCs过度增生,促进其凋亡。

Objective:To investigate the effects of Luteolin contained serum on the high glucose-induced apoptosis of glomerular mesangial cells(GMCs)and the signal pathway of adenosine monophosphate-activated protein kinase(AMPK)/silent information regulator 1(SIRT1)/peroxidase proliferator-activated receptor gamma coactivator 1α(PGC-1α).Methods:Healthy SPF male SD rats were divided into normal group,Luteolin low(1.2 g/kg),medium(2.4 g/kg),high(3.6 g/kg)dose groups and Rosiglitazone positive group(0.36 mg/kg),with 10 rats in each group,all rats were given the corresponding dose of drugs by gavage,once a day,after 7 days,blood was taken from abdominal aorta to prepare the serum containing drugs.The GMCs of SV40 MES13 mice were divided into normal(control)group(10%normal serum),high glucose model(normal)group(30 mmoL/mL glucose+10%normal serum),Luteolin low(30 mmoL/mL glucose+10%low dose serum),medium(30 mmoL/mL glucose+10%middle dose serum),high(30 mmoL/mL glucose+10%high dose serum)dose groups,positive containing serum group(30 mmoL/mL glucose+10%Rosiglitazone group).The cells in each group were cultured with corresponding dose of drugs for 24 hours,the survival rate and apoptosis rate of GMCs in each group were detected by MTT method and flow cytometry;and Western Blot was used to detect the expressions of AMPK/SIRT-1/PGC-1α,apoptosis associated protein fatty acid synthetase(FAS)and B-lymphoma-2 gene(Bcl-2).Results:Compared with the control group,the GMCs cell density,cytoplasmic microfilament like structure,nuclear particle deposition and other HE staining pathological morphology,survival rate,Bcl-2 protein expression in the model group were all increased(P<0.05),the apoptotic rate,the protein expressions of p-AMPK/AMPK,SIRT-1,PGC-1αand Fas were all decreased(P<0.05).Compared with the model group,the cell density,cytoplasmic microfilament like structure,nuclear particle deposition and other HE staining pathological morphology,survival rate,Bcl-2 protein expression in the low,medium and high dose Luteolin contained serum groups and the positive control group were all decreased(P<0.05),the apoptotic rate,the protein expressions of p-AMPK/AMPK,SIRT-1,PGC-1αand Fas were all increased(P<0.05),and Luteolin contained serum groups showed dose-dependent.There was no difference between the positive control group and high dose Luteolin contained serum group(P>0.05).Conclusions:Luteolin contained serum may inhibit the excessive proliferation of high glucose-induced GMCss and promote its apoptosis by activating the expressions of AMPK/SIRT-1/PGC-1αprotein.