Conclusions
PER3 can decrease the PA-induced oxidative stress and inflammatory factor secretion via inhibiting the lipogenesis in podocytes. 目的: 高脂诱导足细胞损伤是导致肥胖相关性肾病(obesity related glomerulopathy，ORG)的重要因素之一，但机制并不明确。本研究探讨昼夜节律时钟基因3(period circadian clock 3，PER3)在抑制棕榈酸(palmitic acid，PA)诱导的小鼠足细胞氧化应激及炎症因子分泌中的作用和机制。方法: 正常及高脂饮食喂养C57BL/6J小鼠16周，取小鼠肾组织，采用蛋白印迹法检测正常体重组和肥胖组PER3的表达；以不同体积浓度(0、50、150和300 μmol/L)PA分别干预足细胞48 h，检测各浓度组PER3 mRNA及蛋白质的表达；以150 μmol/L PA分别干预足细胞0、24、36及48 h，检测各时间点PER3 mRNA及蛋白质的表达；在足细胞中转染腺病毒(adenovirus，Ad)-PER3或小干扰RNA(small interfering RNA，siRNA)-PER3后48 h，再以150 μmol/L PA干预48 h，检测PA组、Ad-PER3+PA组和siRNA-PER3+PA组足细胞中三酰甘油(triglyceride，TG)的含量；利用RNA测序(RNA sequencing, RNA-seq)检测siRNA-PER3组和siRNA-对照组差异基因的表达；在足细胞中转染Ad-PER3或Ad-对照48 h后再以150 μmol/L PA干预48 h，检测Ad-PER3+PA组和Ad-对照+PA组肾病蛋白(nephrin)、足蛋白(podocin)、足细胞标记蛋白(podocalyxin)、肾小球足突细胞膜黏蛋白(podoplanin)、超氧化物歧化酶1(superoxide dismutase 1，SOD1)、谷胱甘肽过氧化物酶1(glutathione peroxidase 1，GPX1)、过氧化氢酶(catalase，CAT)的表达，以及足细胞内丙二醛(malondialdehyde，MDA)、谷胱甘肽(glutathione，GSH)、肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α）、白细胞介素-6(interleukin- 6，IL-6)、白细胞介素-1β(interleukin-1β，IL-1β)及白细胞介素-2 (interleukin-2，IL-2)的含量。结果: 在肥胖组小鼠肾组织中，PER3的表达较正常体重组小鼠下调(P<0.05)；150 μmol/L PA干预足细胞48 h较0、24、36 h PER3 mRNA表达均显著下调(均P<0.01)；与PA组相比，Ad-PER3+PA组足细胞内TG含量显著减少，相反，siRNA-PER3+PA组足细胞内TG的含量增加(均P<0.05)；在足细胞转染siRNA-PER3后，siRNA-PER3组较siRNA-对照组差异基因在氧化磷酸化、细胞外基质受体的相互作用、TNF-α信号通路、游离脂肪酸代谢及游离脂肪酸降解通路中富集(均P<0.05)；Ad-PER3+PA组在足细胞标志基因(nephrin、podocin、podocalyxin和podoplanin)、足细胞内的氧化应激(SOD1、GPX1、CAT和GSH)及炎症因子(TNF-α、IL-6、IL-1β和IL-2)的分泌较PA组均显著下调(均P<0.05)。结论: PER3可通过抑制足细胞内的脂质合成而减少PA诱导的氧化应激及炎症因子分泌。.
Methods
The C57BL/6J mice were fed with chow and high-fat diet for 16 weeks. The PER3 expression in kidney tissues were detected in the normal body weight group and the obesity group. The PER3 mRNA and protein expression were detected after the podocytes were induced with different concentrations (0, 50, 150 and 300 μmol/L) of PA for 48 h. The PER3 mRNA and protein expression were detected after the podocytes were induced with 150 μmol/L PA for 0, 24, 36, and 48 h. Triglyceride (TG) levels were examined in the PA group, the adenovirus (ad)-PER3+PA group, and the siRNA-PER+PA group after the podocytes were transfected by Ad-PER3 or small interfering RNA (siRNA)-PER3 for 48 h and subsequently were induced with 150 μmol/L PA for 48 h. The differential gene expression was detected using RNA sequencing (RNA-seq) after podocytes were transfected by siRNA-PER3 (siRNA-PER3 group) and siRNA-control (siRNA-control group), respectively. The mRNA levels of nephrin, podocin, podocalyxin, podoplanin, superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), catalase (CAT), and the levels of malondialdehyde (MDA), glutathione (GSH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and interleukin-2 (IL-2) were detected after podocytes were transfected with Ad-PER3 or Ad-control for 48 h and then they were induced by 150 μmol/L PA for 48 h.
Results
The PER3 was down-regulated in the obesity group compared with the normal body weight group (P<0.05), and the PER3 was significantly down-regulated after the podocytes were treated with 150 μmol/L for 48 h compared with 0, 24, and 36 h (all P<0.01). The TG contents were significantly decreased in the Ad-PER3+PA group compared with the PA group (P<0.05). On the contrary, TG contents were increased in the siRNA-PER3+PA group compared with the PA group (P<0.05). The RNA-seq results showed that: compared with the siRNA-control group, the differential genes in the siRNA-PER3 group were enriched in different pathways including oxidative phosphorylation, TNF signaling pathway, extracellular matrix receptor interaction, fatty acid metabolism, and fatty acid degradation (all P<0.05). The podocyte marker genes (nephrin, podocin, podocalyxin and podoplanin), oxidative stress (SOD1, GPX1, CAT and GSH), and inflammation factors (TNF-α, IL-6, IL-1β and IL-2) were significantly down-regulated in the Ad-PER3+PA group compared with the PA group (all P<0.05). Conclusions: PER3 can decrease the PA-induced oxidative stress and inflammatory factor secretion via inhibiting the lipogenesis in podocytes. 目的: 高脂诱导足细胞损伤是导致肥胖相关性肾病(obesity related glomerulopathy，ORG)的重要因素之一，但机制并不明确。本研究探讨昼夜节律时钟基因3(period circadian clock 3，PER3)在抑制棕榈酸(palmitic acid，PA)诱导的小鼠足细胞氧化应激及炎症因子分泌中的作用和机制。方法: 正常及高脂饮食喂养C57BL/6J小鼠16周，取小鼠肾组织，采用蛋白印迹法检测正常体重组和肥胖组PER3的表达；以不同体积浓度(0、50、150和300 μmol/L)PA分别干预足细胞48 h，检测各浓度组PER3 mRNA及蛋白质的表达；以150 μmol/L PA分别干预足细胞0、24、36及48 h，检测各时间点PER3 mRNA及蛋白质的表达；在足细胞中转染腺病毒(adenovirus，Ad)-PER3或小干扰RNA(small interfering RNA，siRNA)-PER3后48 h，再以150 μmol/L PA干预48 h，检测PA组、Ad-PER3+PA组和siRNA-PER3+PA组足细胞中三酰甘油(triglyceride，TG)的含量；利用RNA测序(RNA sequencing, RNA-seq)检测siRNA-PER3组和siRNA-对照组差异基因的表达；在足细胞中转染Ad-PER3或Ad-对照48 h后再以150 μmol/L PA干预48 h，检测Ad-PER3+PA组和Ad-对照+PA组肾病蛋白(nephrin)、足蛋白(podocin)、足细胞标记蛋白(podocalyxin)、肾小球足突细胞膜黏蛋白(podoplanin)、超氧化物歧化酶1(superoxide dismutase 1，SOD1)、谷胱甘肽过氧化物酶1(glutathione peroxidase 1，GPX1)、过氧化氢酶(catalase，CAT)的表达，以及足细胞内丙二醛(malondialdehyde，MDA)、谷胱甘肽(glutathione，GSH)、肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α）、白细胞介素-6(interleukin- 6，IL-6)、白细胞介素-1β(interleukin-1β，IL-1β)及白细胞介素-2 (interleukin-2，IL-2)的含量。结果: 在肥胖组小鼠肾组织中，PER3的表达较正常体重组小鼠下调(P<0.05)；150 μmol/L PA干预足细胞48 h较0、24、36 h PER3 mRNA表达均显著下调(均P<0.01)；与PA组相比，Ad-PER3+PA组足细胞内TG含量显著减少，相反，siRNA-PER3+PA组足细胞内TG的含量增加(均P<0.05)；在足细胞转染siRNA-PER3后，siRNA-PER3组较siRNA-对照组差异基因在氧化磷酸化、细胞外基质受体的相互作用、TNF-α信号通路、游离脂肪酸代谢及游离脂肪酸降解通路中富集(均P<0.05)；Ad-PER3+PA组在足细胞标志基因(nephrin、podocin、podocalyxin和podoplanin)、足细胞内的氧化应激(SOD1、GPX1、CAT和GSH)及炎症因子(TNF-α、IL-6、IL-1β和IL-2)的分泌较PA组均显著下调(均P<0.05)。结论: PER3可通过抑制足细胞内的脂质合成而减少PA诱导的氧化应激及炎症因子分泌。.