摘要
目的研究富含亮氨酸重复序列的普列克底物蛋白同源结构域蛋白磷酸酶1(PHLPP1)在糖尿病肾病(DN)肾组织的表达及其对足细胞自噬、凋亡的影响并初步探究其相关作用机制。方法采用免疫组织化学检测DN肾组织及非糖尿病肾组织PHLPP1表达,免疫荧光组织化学染色检测肾病蛋白(nephrin)、 PHLPP1的共表达以确定PHLPP1在足细胞的定位;在正常葡萄糖(NG)及高糖(HG)培养液中培养足细胞,实时荧光定量PCR检测细胞PHLPP1 mRNA表达。采用脂质体瞬时转染技术将靶向沉默PHLPP1的小干扰RNA(si-PHLPP1)转染入足细胞,实时定量PCR检测转染效率;按足细胞处理方式的不同将细胞分为NG组(正常葡萄糖浓度的培养液进行培养的足细胞)、 HG组(高糖培养液培养的足细胞)、 HG联合si-PHLPP1组(高糖培养液培养转染si-PHLPP1后的足细胞组)、羟氯喹(HCQ)处理的HG组(用自噬抑制剂HCQ与HG培养液联合处理的足细胞)。透射电镜观察各组足细胞中自噬小体的形成, Western blot法检测微管相关蛋白1轻涟3(LC3)、 P62、磷脂酰肌醇3激酶(PI3K)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化的mTOR(p-mTOR)、裂解型胱天蛋白酶3(c-caspase-3)、蛋白激酶B (AKT)及磷酸化的AKT(p-AKT)蛋白表达,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术检测细胞凋亡。结果 PHLPP1在DN患者肾组织中高表达,且主要表达于肾小球足细胞内。HG培养液能够促进足细胞中PHLPP1 mRNA表达,并具有时间依赖性;与NG组相比, HG组、 HG联合si-PHLPP1组及HG联合HCQ组的足细胞自噬水平、 PI3K蛋白表达与mTOR的磷酸化水平均显著减低,细胞凋亡率、 c-caspase-3蛋白表达水平均显著增加,但HG联合si-PHLPP1组细胞的AKT磷酸化水平明显升高,其余两组AKT磷酸化水平显著降低;与HG组相比, HG联合si-PHLPP1组细胞的凋亡率、c-caspase-3蛋白表达均明显降低,而自噬水平, PI3K蛋白表达与mTOR和AKT蛋白的磷酸化水平均显著增加, HG联合HCQ组细胞的自噬水平明显降低,凋亡率与c-caspase-3蛋白表达均显著增加,其他指标无明显变化。结论 PHLPP1在DN肾组织显著高表达,下调足细胞PHLPP1的表达通过激活PI3K/AKT/mTOR通路促进足细胞自噬水平,减少足细胞的凋亡。
Objective To investigate the expression of pleckstrin homology(PH) domain leucine-rich repeats protein phosphatase 1(PHLPP1) in renal tissue of patients with diabetic nephropathy(DN) and its effect on podocyte autophagy and apoptosis, and to explore its related mechanism. Methods Immunohistochemistry was used to detect PHLPP1 expression in renal tissue of patients with DN and non-diabetes, and immunofluorescence histochemical staining was used to detect the co-expression of nephrin and PHLPP1 to determine the localization of PHLPP1 in podocytes. Human glomerular podocyte cell line was cultured in normal glucose(NG) and high glucose(HG) media. The expression of PHLPP1 mRNA was detected by real-time quantitative PCR. Small interfering RNA of PHLPP1(si-PHLPP1) targeting down-regulation of PHLPP1 wastransfected into podocytes by Lipofectamine transient transfection technology, and the transfection efficiency was assessed by real-time PCR. According to the different treatment of podocytes, the cells were divided into NG group(podocytes cultured with normal glucose media), HG group(podocytes cultured with high glucose medium), HG combined with si-PHLPP1 group(podocytes transfected with si-PHLPP1 were cultured in high glucose medium), and HG combined with HCQ group(podocytes treated with hydroxychloroquine and high glucose medium). The formation of autophagic vesicles was observed by transmission electron microscope, and the protein expression levels of LC3, P62, PI3K, mTOR, p-mTOR, cleaved caspase 3(c-caspase-3), AKT, p-AKT were detected by Western blotting. Annexin V-FITC/PI staining combined with flow cytometry was used to detect apoptosis. Results PHLPP1 was highly expressed in the renal tissue of patients with DN, and it was mainly expressed in the glomerular podocyte. The HG culture medium could promote the expression of PHLPP1 mRNA in podocytes in a time-dependent manner. Compared with NG group, the autophagy level of podocytes, the expression of PI3K and the phosphorylation level of mTOR in the HG group, HG combined with si-PHLPP1 group and HG combined with HCQ group were significantly reduced;the apoptosis rate and c-caspase-3 protein expression level were significantly enhanced;the phosphorylation level of AKT in the HG combined with si-PHLPP1 group significantly increased, but it in the other two groups significantly decreased. Compared with HG group, the apoptosis rate and c-caspase-3 protein expression in the HG combined with si-PHLPP1 group were significantly reduced, while autophagy level, PI3K protein expression and phosphorylation level of mTOR and Akt protein were significantly elevated. The autophagy level of HG combined with HCQ group was significantly inhibited, the apoptosis rate and c-caspase-3 protein expression were significantly raised, and other indicators showed no significant changes. Conclusion PHLPP1 is highly expressed in renal tissue of patients with DN, and the down-regulated expression of PHLPP1 in podocytes can promote the autophagy of podocytes and reduced the apoptosis of podocytes by activating PI3K/AKT/mTOR pathway.
作者
蔡根深
张晶
王汝朋
CAI Genshen;ZHANG Jing;WANG Rupeng(Department of General Medicine,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2021年第1期8-15,共8页
Chinese Journal of Cellular and Molecular Immunology
基金
北京市科研课题(2016-q24)。