曹东维,韩文贝,何劲松,赵敏,蒋春明,张庆燕,万骋,刘晶,冯媛,金波,杨波,朱大龙,韩晓
CAO Dong-wei,HAN Wen-bei,HE Jin-song,ZHAO Min,JIANG Chun-ming,ZHANG Qing-yan,WAN Cheng,LIU Jing,FENG Yuan,JIN Bo,YANG Bo,ZHU Da-long,HAN Xiao

• 1:南京医科大学鼓楼临床医学院肾内科
• 2:南京大学医学院附属南京鼓楼医院肾内科
• 3:南京中医药大学中西医结合鼓楼临床医学院中医科
• 4:上海中医药大学附属龙华医院皮肤科

摘要(Abstract)：

探讨茶多酚(tea polyphenols,TP)在体外高糖环境下延缓人肾小球系膜细胞(human glomerular mesangial cells,HGMCs)衰老的作用及可能的机制。在体外培养HGMCs,分为正常组(normal group,N,5.5 mmol·L~(-1)葡萄糖)、甘露醇组(mannitol group,MNT,5.5 mmol·L~(-1)葡萄糖+24.5 mmol·L~(-1)甘露醇)、高糖组(high dose of D-glucose group,HG,30 mmol·L~(-1)葡萄糖)、低剂量TP组(low dose of TP group,L-TP,30 mmol·L~(-1)葡萄糖+5 mg·L~(-1) TP)及高剂量TP组(high dose of TP group,H-TP,30 mmol·L~(-1)葡萄糖+20 mg·L~(-1) TP),分别在37℃,5%CO_2条件下培养,干预72 h后,首先观察TP对HGMCs形态的影响;其次,检测细胞周期、衰老相关半乳糖苷酶(senescence-associated-β-galactosidase,SA-β-gal)染色阳性率、端粒长度;最后,检测p53-p21-Rb信号通路中关键信号分子p53,p21,Rb的蛋白表达水平及p-STAT3,miR-126的表达水平。结果表明,高糖能诱导HGMCs衰老,不仅表现为细胞周期阻滞于G_1期、SA-β-gal染色阳性率升高、端粒长度缩短,还表现为p53,p21,Rb蛋白表达水平升高和p53-p21-Rb信号通路激活。L-TP能延缓HGMCs衰老,不仅表现为HGMCs细胞周期G_1期阻滞的改善、SA-β-gal染色阳性率的下降、端粒长度的延长,还表现为p53,p21,Rb蛋白表达水平的下降,端粒-p53-p21-Rb信号通路活性的抑制。此外,高糖诱导HGMCs p-STAT3表达水平上调、miR-126表达水平下调,而L-TP可使这些变化得到改善。总之,高糖能激活端粒-p53-p21-Rb信号通路而诱导HGMCs衰老;L-TP能调节STAT3/miR-126表达水平,抑制端粒-p53-p21-Rb信号通路活性而延缓高糖诱导的HGMCs衰老。这些发现为临床上防治糖尿病肾病相关的肾脏细胞衰老提供了有效的干预措施。
The aim of this paper was to explore the effects and possible mechanisms in vitro of tea polyphenols(TP) delaying human glomerular mesangial cells(HGMCs) senescence induced by high glucose(HG). HGMCs were cultured in vitro and divided into the normal group(N, 5.5 mmol·L~(-1) glucose), the mannitol group(MNT, 5.5 mmol·L~(-1 )glucose plus 24.5 mmol·L~(-1 )mannitol), the high dose of D-glucose group(HG, 30 mmol·L~(-1) glucose), the low dose of TP group(L-TP, 30 mmol·L~(-1) glucose plus 5 mg·L~(-1) TP) and the high dose of TP group(H-TP, 30 mmol·L~(-1 )glucose plus 20 mg·L~(-1) TP), which were cultured in 5% CO_2 at 37 ℃, respectively. Firstly, the effects of TP on the cell morphology of HGMCs were observed after 72 h-intervention. Secondly, the cell cycle, the positive rate of senescence-associated-β-galactosidase(SA-β-gal) staining and the telomere length were detected, respectively. Finally, the protein expressions of p53, p21 and Rb in the p53-p21-Rb signaling pathway were investigated, respectively. And the expressions of p-STAT3 and miR-126 were examined severally. The results indicated that HG not only arrested the cell cycle in G_1 phase but also increased the positive rate of SA-β-gal staining, and shortened the telomere length. HG led to the protein over-expressions of p53, p21 and Rb and HGMCs senescence by activating the p53-p21-Rb signaling pathway. In addition, L-TP delayed HGMCs senescence by improving the cell cycle G_1 arrest, reducing SA-β-gal staining positive rate and lengthening the telomere length. L-TP reduced the protein over-expressions of p53, P21 and Rb induced by HG and inhibited the telomere-p53-p21-Rb signaling pathway. Moreover, the expression of p-STAT3 was increased and the expression of miR-126 was decreased in HGMCs induced by HG. L-TP reduced the expression of p-STAT3 and increased the expression of miR-126 in HGMCs. In conclusion, HG could induce HGMCs senescence by activating the telomere-p53-p21-Rb signaling pathway in vitro. L-TP could delay HGMCs senescence through regulating STAT3/miR-126 expressions and inhibiting the telomere-p53-p21-Rb signaling pathway activation. These findings could provide the effective interventions in clinic for preventing and treating renal cell senescence in diabetic kidney disease.

关键词(KeyWords)： 糖尿病肾病;茶多酚;系膜细胞衰老;STAT3;miR-126;端粒
diabetic kidney disease;tea polyphenols;human glomerular mesangial cells senescence;STAT3;miR-126;telomere

Abstract:

Keywords:

基金项目(Foundation): 南京市医学科技发展项目(YKK15056,YKK16097);; 国家自然科学基金面上项目(81473684)

作者(Author): 曹东维,韩文贝,何劲松,赵敏,蒋春明,张庆燕,万骋,刘晶,冯媛,金波,杨波,朱大龙,韩晓
CAO Dong-wei,HAN Wen-bei,HE Jin-song,ZHAO Min,JIANG Chun-ming,ZHANG Qing-yan,WAN Cheng,LIU Jing,FENG Yuan,JIN Bo,YANG Bo,ZHU Da-long,HAN Xiao

DOI: 10.19540/j.cnki.cjcmm.20181031.003

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