基于自噬信号通路探究褐藻多糖硫酸酯改善人近端肾小管上皮细胞衰老的分子机制

Exploring molecular mechanisms of fucoidan in improving human proximal renal tubular epithelial cells aging by targeting autophagy signaling pathways

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-associatedβ-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(25μg·mL^(-1))or D-gal(75 mmol·L^(-1))+FPS(50μg·mL^(-1))or D-gal(75 mmol·L^(-1))+VE(50μg·mL^(-1)).After the treatment for 24 h,firstly,the effects of D-gal on senescence-associatedβ-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.