房其军;刘建璟;万毅刚;刘不悔;涂玥;吴薇;刘莹露;王文文;王美子;YEE Hong-yun;袁灿灿;CHONG Fee-lan;
FANG Qi-jun;LIU Jian-jing;WAN Yi-gang;LIU Bu-hui;TU Yue;WU Wei;LIU Ying-lu;WANG Wen-wen;WANG Mei-zi;YEE Hong-yun;YUAN Can-can;CHONG Fee-lan;Department of Traditional Chinese Medicine, Nanjing Drum Tower Hospital Clinical College of Chinese Medicine and Western Medicine, Nanjing University of Chinese Medicine;Department of Nephrology, Jiangsu Province Hospital on Integration of Chinese and Western Medicine;Department of Traditional Chinese Medicine, Nanjing Drum Tower Hospital, the Affiliated Hos

• 1:南京中医药大学中西医结合鼓楼临床医学院中医科
• 2:江苏省中西医结合医院肾内科
• 3:南京大学医学院附属鼓楼医院中医科
• 4:南京中医药大学附属医院肾内科

摘要(Abstract)：

褐藻多糖硫酸酯(fucoidan,FPS)是临床上治疗慢性肾衰竭中成药海昆肾喜胶囊的有效成分,具有潜在的抗衰老作用。然而,FPS是否能改善肾脏衰老,尤其是对于其抗衰老的分子机制,目前仍不清楚。将体外培养的人近端肾小管上皮细胞(HK-2)分为正常组(normal group,N)、模型组(D-gal model group,D)、低剂量FPS组(low dose of FPS,L-FPS)、高剂量FPS组(high dose of FPS,H-FPS)以及维生素E组(vitamin E,VE),分别进行不同的干预。N组加入1%胎牛血清(fetal bovine serum,FBS)1 mL;D组加入75 mmol·L~(-1) D-gal;L-FPS组加入75 mmol·L~(-1) D-gal+25μg·mL~(-1) FPS;H-FPS组加入75 mmol·L~(-1) D-gal+50μg·mL~(-1) FPS;VE组加入75 mmol·L~(-1) D-gal+50μg·mL~(-1) VE。在干预后24 h,首先,观察D-gal对HK-2细胞β-半乳糖苷酶(senescence-associated β-galactosidase,SA-β-gal)染色特征以及klotho,P53,P21蛋白表达水平和腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)-不协调的51类激酶1(uncoordinated 51-like kinase 1,ULK1)信号通路活性的影响;其次,观察FPS和VE对D-gal诱导的HK-2细胞SA-β-gal染色特征以及klotho,P53,P21蛋白表达水平的影响;最后,观察FPS和VE对D-gal诱导的HK-2细胞哺乳动物同族物微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)蛋白表达水平以及AMPK-ULK1信号通路活性的影响。结果表明,对于HK-2细胞,75 mmol·L~(-1)的D-gal能安全而有效地诱导细胞SA-β-gal染色特征和klotho,P53,P21蛋白表达水平的变化,也就是引发细胞衰老;而FPS和VE都能改善D-gal诱导的细胞SA-β-gal染色特征以及klotho,P21,P53蛋白表达水平的变化,也就是拮抗细胞衰老,这里,两者的作用是类似的;D-gal不仅能诱导细胞衰老,还能促进LC3Ⅱ,磷酸化AMPK(phosphorylated-AMPK,p-AMPK)以及磷酸化ULK1(phosphorylated-ULK1,p-ULK1)蛋白高表达,激活自噬相关AMPK-ULK1信号通路;而FPS和VE都能改善D-gal诱导的细胞LC3Ⅱ,p-AMPK以及p-ULK1蛋白表达水平的变化,也就是抑制自噬相关AMPK-ULK1信号通路活性。总之,对于D-gal诱导的人近端肾小管上皮细胞衰老模型,FPS与VE相似,能改善肾脏细胞衰老,其分子机制可能与抑制自噬相关AMPK-ULK1信号通路活性有关。这一发现为FPS抗肾脏衰老提供了初步的药理学证据。
Fucoidan(FPS) is an effective component of the Chinese patent medicine named Haikun Shenxi, which treats schronic renal failure in clinics, and has the potential anti-aging effects. However, it is still unclear whether FPS can improve renal aging, especially the molecular mechanism of its anti-aging. The human proximal renal tubular epithelial cells(HK-2) in vitro were divided into normal group(N), D-gal model group(D), low dose of FPS group(L-FPS), high dose of FPS group(H-FPS) and vitamin E group(VE), and treated by the different measures, respectively. More specifically, the HK-2 cells in each group were separately treated by 1 mL of 1% fetal bovine serum(FBS) or D-galactose(D-gal, 75 mmol·L~(-1)) or D-gal(75 mmol·L~(-1))+FPS(25 μg·mL~(-1)) or D-gal(75 mmol·L~(-1))+FPS(50 μg·mL~(-1)) or D-gal(75 mmol·L~(-1))+VE(50 μg·mL~(-1)). After the treatment for 24 h, firstly, the effects of D-gal on senescence-associated β-galactosidase(SA-β-gal) staining characteristics and klotho, P53 and P21 protein expression le-vels, as well as adenosine monophosphate activated protein kinase(AMPK)-uncoordinated 51-like kinase 1(ULK1) signaling pathway activation in the HK-2 cells were detected, respectively. Secondly, the effects of FPS and VE on SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels in the HK-2 cells exposed to D-gal were investigated, respectively. Finally, the effects of FPS and VE on microtubule-associated protein 1 light chain 3(LC3) protein expression level and AMPK-ULK1 signaling pathway activation in the HK-2 cells exposed to D-gal were examined severally. The results indicated that, for the HK-2 cells, the dose of 75 mmol·L~(-1) D-gal could induce the changes of SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels. That is causing cells aging. FPS and VE could both ameliorate the changes of SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels in the HK-2 cells exposed to D-gal. That is anti-cells aging, here, the functions of FPS and VE are similar. D-gal could not only induce cell aging but also increase LC3Ⅱ, phosphorylated-AMPK(p-AMPK) and phosphorylated-ULK1(p-ULK1) protein expressions, and activate autophagy-related AMPK-ULK1 signaling pathway. FPS and VE could both improve the changes of LC3Ⅱ, p-AMPK and p-ULK1 protein expression levels in the HK-2 cells exposed to D-gal. That is inhibiting autophagy-related AMPK-ULK1 signaling pathway activation. On the whole, for the human proximal renal tubular epithelial cells aging models induced by D-gal, FPS similar to VE, can ameliorate renal cells aging by possibly inhibiting autophagy-related AMPK-ULK1 signaling pathway activation. This finding provides the preliminary pharmacologic evidences for FPS protecting against renal aging.

关键词(KeyWords)： 衰老;褐藻多糖硫酸酯;人近端肾小管上皮细胞;自噬;AMPK-ULK1信号通路
aging;fucoidan;human proximal renal tubular epithelial cells;autophagy;AMPK-ULK1 signaling pathway

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金面上项目(81573903);国家自然科学基金青年科学基金项目(81603675);; 江苏省中医药科技发展计划项目(YB201937);; 南京市医学科技发展基金项目(QRX17042);; 南京市名中医专家工作室建设项目

作者(Author): 房其军;刘建璟;万毅刚;刘不悔;涂玥;吴薇;刘莹露;王文文;王美子;YEE Hong-yun;袁灿灿;CHONG Fee-lan;
FANG Qi-jun;LIU Jian-jing;WAN Yi-gang;LIU Bu-hui;TU Yue;WU Wei;LIU Ying-lu;WANG Wen-wen;WANG Mei-zi;YEE Hong-yun;YUAN Can-can;CHONG Fee-lan;Department of Traditional Chinese Medicine, Nanjing Drum Tower Hospital Clinical College of Chinese Medicine and Western Medicine, Nanjing University of Chinese Medicine;Department of Nephrology, Jiangsu Province Hospital on Integration of Chinese and Western Medicine;Department of Traditional Chinese Medicine, Nanjing Drum Tower Hospital, the Affiliated Hos

Email:

DOI: 10.19540/j.cnki.cjcmm.20200709.401

参考文献(References)：

• [1] GORGOULIS V,ADAMS P D,ALIMONTI A,et al.Cellular senescence:defining a path forward [J].Cell,2019,179(4):813.
• [2] AHADI S,ZHOU W,SCHüSSLER-FIORENZA R S M,et al.Personal aging markers and ageotypes revealed by deep longitudinal profiling [J].Nat Med,2020,26(1):83.
• [3] STURMLECHNER I,DURIK M,SIEBEN C J,et al.Cellular senescence in renal ageing and disease [J].Nat Rev Nephrol,2017,13(2):77.
• [4] CHILDS B G,BUSSIAN T J,BAKER D J.Cellular identification and quantification of senescence-associated β-galactosidase activity in vivo[J].Methods Mol Biol,2019,1896:31.
• [5] ZOU D,WU W,HE Y,et al.The role of klotho in chronic kidney disease [J].BMC Nephrol,2018,19(1):285.
• [6] KIM Y Y,JEE H J,UM J H,et al.Cooperation between p21 and Akt is required for p53-dependent cellular senescence[J].Aging Cell,2017,16(5):1094.
• [7] 刘不悔,何伟明,万毅刚,等.虫草菌丝抑制自噬相关AMPK/ULK1信号活性改善肾小管上皮细胞衰老的分子机制[J].中国中药杂志,2019,44(6):1258.
• [8] LIU B,TU Y,HE W,et al.Hyperoside attenuates renal aging and injury induced by D-galactose via inhibiting AMPK-ULK1 signaling-mediated autophagy [J].Aging(Albany NY),2018,10(12):4197.
• [9] 刘不悔,顾一煌,涂玥,等.衰老的分子调控机制及中药的干预作用[J].中国中药杂志,2017,42(16):3065.
• [10] LIN T A,WU V C,WANG C Y.Autophagy in chronic kidney diseases [J].Cells,2019,8(1):61.
• [11] KIMURA T,ISAKA Y,YOSHIMORI T.Autophagy and kidney inflammation[J].Autophagy,2017,13(6):997.
• [12] DIKIC I,ELAZAR Z.Mechanism and medical implications of mammalian autophagy[J].Nat Rev Mol Cell Biol,2018,19(6):349.
• [13] BAISANTRY A,BHAYANA S,RONG S,et al.Autophagy induces prosenescent changes in proximal tubular S3 segments [J].J Am Soc Nephrol,2016,27(6):1609.
• [14] JOACHIM J,JEFFERIES H B,RAZI M,et al.Activation of ULK kinase and autophagy by GABARAP trafficking from the centrosome is regulated by WAC and GM130 [J].Mol Cell,2015,60(6):899.
• [15] FITTON J H,STRINGER D N,KARPINIEC S S.Therapies from fucoidan:an update [J].Mar Drugs,2015,13(9):5920.
• [16] WANG X,YI K,ZHAO Y.Fucoidan inhibits amyloid-β-induced toxicity in transgenic Caenorhabditis elegans by reducing the accumulation of amyloid-β and decreasing the production of reactive oxygen species [J].Food Funct,2018,9(1):552.
• [17] SHU B,DUAN W,YAO J,et al.caspase 3 is involved in the apo-ptosis induced by triptolide in HK-2 cells [J].Toxicol In Vitro,2009,23(4):598.
• [18] FISCHER M,QUAAS M,STEINER L,et al.The p53-p21-DREAM-CDE/CHR pathway regulates G2/M cell cycle genes [J].Nucleic Acids Res,2016,44(1):164.
• [19] HODGSON R,KENNEDY B K,MASLIAH E,et al.Aging:the-rapeutics for a healthy future [J].Neurosci Biobehav Rev,2020,108:453.
• [20] PARTRIDGE L,FUENTEALBA M,KENNEDY B K.The quest to slow ageing through drug discovery[J].Nat Rev Drug Discov,2020,doi:10.1038/s41573-020-0067-7.
• [21] 邢玉瑞.中医浊毒概念问题探讨[J].中医杂志,2017,58(14):1171.
• [22] 黄福发,黄毅,黄俊,等.浅谈血瘀致衰论 [J].中国中医药现代远程教育,2012,10(24):128.
• [23] RUBINSZTEIN D C,MARINO G,KROEMER G.Autophagy and aging [J].Cell,2011,146(5):682.
• [24] WANG Z,CHOI M E.Autophagy in kidney health and disease [J].Antioxid Redox Signal,2014,20(3):519.
• [25] KIMURA T,TAKABATAKE Y,TAKAHASHI A,et al.Autopha-gy protects the proximal tubule from degeneration and acute ischemic injury [J].J Am Soc Nephrol,2011,22(5):902.
• [26] WANG C,YU Z,SHI X,et al.Triclosan enhances the clearing of pathogenic intracellular salmonella or Candida albicans but disturbs the intestinal microbiota through mTOR-independent autophagy [J].Front Cell Infect Microbiol,2018,8:49.
• [27] PARK J S,CHOI H I,KIM D H,et al.Alpha-lipoic acid attenua-tes p-cresyl sulfate-induced renal tubular injury through suppression of apoptosis and autophagy in human proximal tubular epithelial cells [J].Biomed Pharmacother,2019,112:108679.
• [28] RECKELHOFF J F,KANJI V,RACUSEN L C,et al.Vitamin E ameliorates enhanced renal lipid peroxidation and accumulation of F2-isoprostanes in aging kidneys [J].Am J Physiol,1998,74(3):R767.
• [29] 邓蓉,谢丽芬,成杏芳,等.褐藻多糖硫酸酯减轻尿酸性肾病大鼠肾小管上皮细胞损伤的作用机制 [J].暨南大学学报,2018,39(3):208.