Abstract
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。
Keywords: PER3, 棕榈酸, 足细胞, 氧化应激, 炎症因子
Abstract
Objective
High fat-induced podocyte injury is one of the important factors leading to obesity related nephropathy (ORG), but the mechanism is not clear. This study aims to explore the mechanism of period circadian clock 3 (PER3) in the oxidative stress and inflammation induced by palmitic acid (PA) in podocytes.
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).
Conclusion
PER3 can decrease the PA-induced oxidative stress and inflammatory factor secretion via inhibiting the lipogenesis in podocytes.
Keywords: period circadian clock 3, palmitic acid, podocytes, oxidative stress, inflammatory factors
肥胖是严重危害人类健康的重大疾病[1]。研究[2]显示：肥胖导致肥胖相关性肾病(obesity related glomerulopathy，ORG)的发生，ORG的发病机制为多因素的共同作用，其中肥胖导致的炎症因子分泌增加、脂毒性、氧化应激、胰岛素抵抗等尤为重要，上述因素导致的肾小球足细胞的受损是ORG发生的病理和生理基础[3-4]。
氧化应激和炎症因子是导致ORG足细胞损伤的重要因素。脂肪组织分泌的炎症因子(如TNF-α、IL-1及IL-6)导致产生过多的活性氧族(reactive oxygen species，ROS)，此外，过多的足细胞内脂质形成可增加还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate，NADPH)的氧化活性，同样导致ROS的产生，过多ROS的产生导致足细胞的增殖、分化等功能障碍[5]。ROS的产生又能激活氧化还原敏感转录因子(尤其是NF-κB)，或启动丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)等炎症通路而进一步促进炎症因子的产生，从而加重炎症反应的过程[6]。
昼夜节律时钟基因3(period circadian clock 3，PER3)是昼夜节律调控系统中的重要分子之一[7]。近年来的研究[8-9]表明：PER3负调控小鼠及人脂肪细胞分化并减少脂质的合成，分子机制包括小鼠PER3蛋白N端1~378氨基酸序列能与脂肪细胞分化的关键核转录因子过氧化物酶体增殖物激活受体γ(peroxisome proliferative activated receptor γ，PPARγ)蛋白相互作用并下调PPARγ表达；PER3还能通过下调Krüppel样因子15(Krüppel like factor 15，Klf15)的表达，抑制脂肪细胞的分化；敲除小鼠PER3导致体重增加[10-11]。
ORG的发病机制目前尚不明确，且昼夜节律调控系统的基因对足细胞的功能影响的研究仍非常少。本研究拟探讨PER3在棕榈酸(palmitic acid，PA)诱导下的足细胞损伤的保护机制，为ORG发病机制的研究提供新的思路及治疗靶点。
1. 材料与方法
1.1. 材料
1.1.1. 动物
6周龄C57BL/6J小鼠(体重15~20 g)购自湖南斯莱克景达实验动物公司，饲养于中南大学湘雅医学院实验动物学部。
1.1.2. 细胞、试剂及仪器
小鼠足细胞MPC5由中南大学湘雅三医院内分泌实验室保存；RPMI 1640培养基购自美国HyClone公司；胎牛血清购自美国Gibco公司；干扰素-γ (interferon-γ，INF-γ)、Triton X-100及PA购自美国Sigma公司；TRIzol试剂盒购自美国Life公司；反转录试剂盒购自美国Promega公司；LipofectamineTM 3000购自美国Invitrogen公司；小鼠siRNA-PER3及siRNA-对照购自广州锐博生物科技有限公司；腺病毒(adenovirus，Ad)-对照及Ad-PER3由上海吉凯基因医学科技股份有限公司构建及包装；三酰甘油(triglyceride，TG)检测试剂盒购自美国Abcam公司(ab65336)；免疫沉淀(immunoprecipitation，IP)细胞裂解液(P0013)、丙二醛(malondialdehyd，MDA)试剂盒及还原型谷胱甘肽(glutathione，GSH)检测试剂盒购自上海碧云天生物技术有限公司；TNF-α、IL-6、IL-2和IL-1β ELISA试剂盒购自美国BD公司；SOD1、PER3及GAPDH购自武汉三鹰生物技术有限公司；GXP1抗体购自美国Santa Cruz公司；高脂饮食(60%脂肪，20%碳水化合物及20%蛋白质购自美国Research Diets公司；real-time PCR仪(Mastercycler® real-time PCR)购自德国Eppendorf公司；Multiskan FC型分光光度仪购自美国Thermo Fisher Scientific公司；Gel Doc XR型凝胶成像分析系统购自美国Bio-Rad 公司。
1.2. 方法
1.2.1. 肥胖小鼠模型的建立
取6周龄C57BL/6J小鼠各5只分别给予正常及高脂饮食饲养16周后，测量体重、血糖及血脂，分为正常体重组及肥胖组，取两组小鼠肾行蛋白质印迹法检测。
1.2.2. 细胞培养
将MPC5细胞培养在含有10%胎牛血清及10 U/mL IFN-γ的RPMI 1640培养基中，培养条件为33 ℃，5% CO2的恒温培养。待细胞密度达70%~80%时，将细胞培养在含10%胎牛血清但不含IFN-γ的RPMI-1640培养基中，在37 ℃下培养14 d以促进MPC5细胞分化为成熟的足细胞。所有细胞实验均在成熟的足细胞中进行。
1.2.3. 细胞转染及PA干预
将Ad-PER3、Ad-对照、siRNA-PER3及siRNA-对照分别转染诱导成熟的足细胞，将细胞分为Ad-PER3组、Ad-对照组、siRNA-PER3组及siRNA-对照组。细胞转染48 h后，再采用150 μmol/L PA干预转染或未转染细胞48 h，细胞被分为未用PA干预的对照组、PA组、Ad-PER3+PA组及siRNA-PER3+PA组。
1.2.4. Real-time PCR检测
以PBS液轻柔冲洗各组细胞3次，每组细胞加用TRIzol 1 mL，根据TRIzol及反转录试剂盒说明书抽提总RNA及反转录成cDNA，用real-time PCR检测各基因的表达。以小鼠GAPDH为内参，采用2-ΔΔCt法计算各组细胞基因mRNA的相对表达[ΔΔCt=(Ct样本1目标基因-Ct样本1 GAPDH基因)-(Ct样本2目标基因- Ct样本2 GAPDH基因)]。引物序列见表1。
表1.
Real-time PCR引物序列
Table 1 Primer sequences of real-time PCR
| 基因 | 引物序列 |
|---|---|
| PER3 | 正向: 5'-AACACGAAGACCGAAACAGAAT-3' |
| 反向: 5'-CTCGGCTGGGAAATACTTTTTCA-3' | |
| Nephrin | 正向: 5'-ATGGGAGCTAAGGAAGCCACA-3' |
| 反向: 5'-CCACACCACAGCTTAACTGTC-3' | |
| Podocalyxin | 正向: 5'-AGTGCCACAACATCAACAGAA-3' |
| 反向: 5'-TGTGAATGGTGTAGGGTTGCT-3' | |
| Podocin | 正向: 5'-GCATCAAGCCCTCTGGATTAG-3' |
| 反向: 5'-AGACGGAGATCAACCTTGTGATA-3' | |
| Podoplanin | 正向: 5'-GTTTTGGGGAGCGTTTGGTTC-3' |
| 反向: 5'-CATTAAGCCCTCCAGTAGCAC-3' | |
| SOD1 | 正向: 5'-AACCAGTTGTGTTGTCAGGAC-3' |
| 反向: 5'-CCACCATGTTTCTTAGAGTGAGG-3' | |
| GPX1 | 正向: 5'-CCACCGTGTATGCCTTCTCC-3' |
| 反向: 5'-AGAGAGACGCGACATTCTCAAT-3' | |
| CAT | 正向: 5'-GGAGGCGGGAACCCAATAG-3' |
| 反向: 5'-GTGTGCCATCTCGTCAGTGAA-3' | |
| GAPDH | 正向: 5'-AGGTCGGTGTGAACGGATTTG-3' |
| 反向:5'- GGGGTCGTTGATGGCAACA-3' |
PER3：昼夜节律时钟基3；Nephrin：肾病蛋白；Podocalyxin：足细胞标记蛋白；Podocin：足蛋白；Podoplanin：肾小球足突细胞膜黏蛋白；SOD1：超氧化物歧化酶1；GPX1：谷胱甘肽过氧化物酶1；CAT：过氧化氢酶；GAPDH：甘油醛-3-磷酸脱氢酶。
1.2.5. 蛋白质印迹法检测
小鼠肾组织在液氮中研磨，各组细胞以PBS洗3遍，均加入IP细胞裂解液，并将裂解液转移至1.5 mL离心管中，在4 ℃下充分裂解后，以10 000×g离心10 min，吸取上清液，测定蛋白浓度。配置8%~12%的分离胶，采用常规方法将蛋白质变性，加样器上样，每孔上样约50 μg，以200 V恒压电泳约45 min，将蛋白转膜至聚偏二氟乙烯膜上，用5%的脱脂牛奶在室温下封闭1 h，加入一抗于4 ℃下孵育过夜，加入二抗在室温下孵育1 h。采用ECL显影、Gel Doc XR凝胶成像分析系统进行条带分析。
1.2.6. RNA测序
Ad-NC及Ad-PER3转染足细胞后48 h，两组细胞用PBS液轻柔清洗3遍，分别加入TRIzol 1 mL，送上海吉凯基因医学科技股份有限公司行RNA测序(RNA sequencing，RNA-seq)。基因本体论(Gene Ontology，GO)分析利用GOseq R软件包，通路分析使用京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)数据库(www.genome.jp/kegg)和基于KEGG直系同源的注释系统(KEGG Orthology Based Annotation System，KOBAS)软件。
1.2.7. 细胞内TG测定
收集约4×105个足细胞，加入含有1%的Triton X-100的PBS液中，在振荡器上充分混匀1 min，冰上裂解30 min，在4 ℃下离心(10 000 g，15 min)，收集上清液后按照TG检测试剂盒说明书操作，用分光光度仪在570 nm波长处检测TG的含量。
1.2.8. 细胞内MDA和GSH含量测定
用PBS液洗涤细胞3次，收集各组细胞，每组加入0.1 mL IP细胞裂解液，以12 000 g离心10 min，收集上清液，按照MDA说明书执行操作。用PBS液洗涤细胞3次，收集各组细胞，加入30 μL的蛋白质去除试剂M溶液，于振荡器上充分混匀，反复在液氮及37 ℃水浴中冻融2次，冰浴5 min，以10 000 g离心10 min，按照GSH试剂盒说明书检测细胞内GSH含量。
1.2.9. 细胞上清液TNF-α、IL-6、IL-2和IL-1β 的检测
收集各组细胞培养基，按照ELISA试剂盒说明书检测培养基中TNF-α、IL-6、IL-2和IL-1β 的含量，用分光光度仪在450 nm波长处测定各指标含量。
1.3. 统计学处理
数据使用GraphPad Prism 8.0软件分析，所有数据采用均数±标准差( ±s)表示，两组间的比较使用t检验，多组间比较采用单因素方差分析ANOVA检验，P<0.05为差异有统计学意义。
2. 结 果
2.1. PER3抑制PA诱导的足细胞内脂质形成
结果显示：肥胖组较正常体重组PER3蛋白的表达显著下调(P<0.05，图1A)；利用不同体积浓度的PA干预小鼠足细胞48 h后，150 μmol/L PA组较0、50及300 μmol/L PA组PER3 mRNA的表达下调(均P<0.05，图1B)；150 μmol/L PA干预足细胞后48 h，PER3 蛋白较0 μmol/L PA组的表达下调(P<0.05，图1C)；150 μmol/L PA干预足细胞后0，12，24，36及48 h，PER3 mRNA及蛋白质的表达随着干预时间延长而逐渐下调(均P<0.05，图1D、1E)；在足细胞中转染Ad-PER3过表达或siRNA-PER3沉默PER3 48 h后， 150 μmol/L PA干预Ad-PER3组、siRNA-PER3组及未转染的细胞48 h，Ad-PER3+PA组足细胞中TG的含量较PA组显著减少，而siRNA PER3+PA组则较PA组增加(均P<0.05，图1F)。
图1.
PER3调控PA诱导的足细胞内脂质的形成
Figure 1 PER3 regulates PA-induced lipogenesis in podocytes
A: PER3 expressions of kidney tissues were detected in the normal body weight group and the obesity group (n=5). 1 to 3 represent 3 different mice. B: PER3 mRNA expression after podocytes were induced with different concentrations of PA for 48 h (n=3). *P<0.05, **P<0.01 vs the 0 μmol/L PA group. C: PER3 protein expressions after the podocytes were induced with 150 μmol/L PA for 48 h (n=3). D and E: PER3 mRNA (D) and protein (E) expressions during podocytes were induced with 150 μmol/L PA for 0, 12, 24, 36, and 48 h (n=3). *P<0.05, **P<0.01 vs the 0 h group. F: Cellular TG levels after podocytes were over-expressed, silenced, or non-transfected by PER3 for 48 h and then were treated with 150 μmol/L PA for 48 h (n=3). *P<0.05 vs the PA group.
2.2. 过表达PER3减少PA诱导的足细胞损伤
与对照组相比，PA组、Ad-PER3+PA组nephrin、podocin、podocalyxin和podoplanin mRNA显著下调，差异均有统计学意义(P<0.05或P<0.01)；与PA组相比，Ad-PER3+PA组和Ad-对照组nephrin、podocin、podocalyxin及podoplanin mRNA的表达均显著上调，差异均有统计学意义(P<0.05或P<0.01；图2)，而对照组较Ad-对照组差异无统计学意义(P>0.05，图2)。
图2.
PER3保护PA诱导的足细胞受损
Figure 2 PER3 protects podocytes from PA induced injury
A: Nephrin; B: Podocin; C: Podocalyxin; D: Podoplanin. The expressions of podocyte markers mRNA after podocytes were over-expressed by Ad-PER3 or Ad-control or non transfected, and then were induced with 150 μmol/L PA for 48 h (n=3). *P<0.05, **P<0.01 vs the control group; †P<0.05, ††P<0.05 vs the PA group.
2.3. RNA-seq检测沉默PER3后的足细胞差异基因的表达
在足细胞中转染siRNA-PER3及siRNA-对照后48 h，RNA-seq测序检测siRNA-PER3组与siRNA-对照组细胞比较差异基因的表达，结果发现共有988个基因差异倍数≥1.0，其中上调基因630个，下调基因358个，在上调的基因中包括炎症因子相关基因(如IL6、IL1β及IL1R2)(表2，3)。通路分析显示：差异表达基因在帕金森病、氧化磷酸化、细胞外基质受体的相互作用及TNF-α信号通路中显著富集，而部分基因在游离脂肪酸代谢、游离脂肪酸降解通路中富集。GO分析显示：差异表达基因在刺激物的反应、阳性调控生物学过程及防御反应的生物过程中显著富集，而在细胞组分中，线粒体、细胞外区部分及细胞外空间等显著富集(图3)。
表2.
上调最显著的10个基因
Table 2 Most significant 10 up-regulated genes
| 基因 | log2倍数改变 | P | Q值 |
|---|---|---|---|
| CSF3 | 10.207 | 2.71E-17 | 1.51E-15 |
| SELE | 9.5494 | 2.18E-34 | 2.35E-32 |
| CXCL15 | 9.0535 | 1.09E-34 | 1.19E-32 |
| TREM1 | 8.6742 | 3.57E-08 | 1.04E-06 |
| IL-6 | 8.3136 | 1.33E-28 | 1.19E-26 |
| KDM5D | 8.0206 | 1.24E-10 | 4.43E-09 |
| OSM | 7.9944 | 1.72E-10 | 6.05E-09 |
| IL-1β | 7.9751 | 3.62E-28 | 3.19E-26 |
| DDX3Y | 7.3666 | 5.83E-20 | 3.70E-18 |
| IL-1R2 | 6.9094 | 4.83E-06 | 0.00010752 |
CSF3：集落刺激因子3；SELE：选择素；CXCL15：趋化因子(C-X-C基序)配体15；TREM1：髓样细胞上表达的触发受体1；IL-6：白细胞介素-6；KDM5D：赖氨酸(K)特异性去甲基酶5D；OSM：抑瘤素M；IL-1β：白细胞介素-1β；DDX3Y：死盒螺旋酶3(Y连锁)；IL-1R2：白细胞介素-1受体2型。
表3.
下调最显著的10个基因
Table 3 Most significant 10 down-regulated genes
| 基因 | log2倍数改变 | P | Q值 |
|---|---|---|---|
| CYP1A2 | -7.9962 | 1.05E-06 | 2.57E-05 |
| SCIN | -7.4671 | 8.77E-17 | 4.73E-15 |
| OGDHL | -7.3645 | 2.92E-12 | 1.20E-10 |
| IGLC2 | -7.3188 | 5.84E-12 | 2.34E-10 |
| ADRB3 | -7.0532 | 2.31E-07 | 6.13E-06 |
| CPN2 | -6.94 | 4.31E-37 | 5.04E-35 |
| TFF3 | -6.8986 | 6.76E-23 | 4.85E-21 |
| MMRN1 | -6.7048 | 3.57E-39 | 4.51E-37 |
| ZDHHC19 | -6.6779 | 4.45E-06 | 1.00E-04 |
| HMGCS2 | -6.3312 | 4.85E-11 | 1.81E-09 |
CYP1A2：细胞色素P450，家族1，亚家族a，多肽2；SCIN：肌切蛋白；OGDHL：氧戊二酸脱氢酶样；IGLC2：免疫球蛋白λ常数2；ADRB3：肾上腺素能受体β3；CPN2：羧肽酶N多肽2；TFF3：三叶因子3，肠道；MMRN1：多聚蛋白1；ZDHHC19：锌指，DHHC结构域含19；HMGCS2：3-羟基-3-甲基戊二酰辅酶A合酶2。
图3.
足细胞沉默PER3后差异基因的表达
Figure 3 Differential gene expression after silencing PER3 in podocytes
A: Pathway enrichment analysis; B: Gene ontology enrichment analysis.
2.4. 过表达PER3抑制PA诱导的足细胞氧化应激
与对照组相比，PA组和Ad-PER3+PA组的SOD1、GPX1和CAT mRNA显著下调，差异均有统计学意义(P<0.05或P<0.01)；与PA组比较，Ad-对照组和Ad-PER3+PA组SOD1、GPX1和CAT mRNA的表达显著上调，差异均有统计学意义(P<0.05或P<0.01，图4A~4C)，而对照组较Ad-对照组并无差异 (P>0.05，图4A~4C)。在足细胞中过表达或沉默PER3后48 h，蛋白质印迹法结果显示：siRNA-PER3组较siRNA-对照组SOD1及GPX1蛋白的表达下调，而Ad-PER3组则较Ad-对照组上调(均P<0.05，图4D)。比色法检测足细胞中转染Ad-PER3后对以 150 μmol/L PA诱导的MDA及GSH的影响，结果发现：与对照组相比，PA组和Ad-PER3+PA组足细胞MPA和GSH显著上调，差异均有统计学意义(P<0.05或P<0.01)；Ad-PER3+PA组足细胞MDA的含量较PA组减少(P<0.05，图4E)，而GSH增加(P<0.05，图4F)；对照组与Ad-对照组MDA及GSH含量比较，差异均无统计学意义(均P>0.05；图4E、4F)。
图4.
PER3减少PA诱导的足细胞氧化应激
Figure 4 PER3 decreases PA-induced oxidative stress in podocytes
A-C: SOD1 (A), GPX1 (B), and CAT (C) mRNA expression after podocytes were over-expressed by Ad-PER3 and Ad-control for 48 h and then were treated with 150 μmol/L PA for 48 h (n=3). D: SOD1 and GPX1 protein expression after over-expression or silencing of PER3 in podocytes for 48h (n=3); E and F: MDA (E) and GSH (F) level after over-expression of PER3 and PA-induced in podocytes for 48 h (n=3). *P<0.05, **P<0.01 vs the control group; †P<0.05, ††P<0.05 vs the PA group.
2.5. 过表达抑制PA诱导的足细胞炎症因子的分泌
与对照组相比，PA组和Ad-PER3+PA组TNF-α、IL-6、IL-1β和IL-2含量显著上调，差异均有统计学意义(P<0.05或P<0.01)；与PA组相比，Ad-对照组和Ad-PER3+PA组TNF-α、IL-6、IL-1β及IL-2的含量均显著下调，差异均有统计学意义(P<0.05或P<0.01)，而对照组较Ad-对照组差异无统计学意义(P>0.05，图5)。
图5.
PER3减少PA诱导的足细胞炎症因子分泌
Figure 5 PER3 inhibits PA induced secretion of inflammatory factors in podocytes
A-D: TNF-α(A), IL-6 (B), IL-1β (C) and IL-2 (D) contents were detected in media after podocytes were over-expressed by PER3 and then were induced with 150 μmol/L PA for 48 h (n=3). *P<0.05, **P<0.01 vs the control group; †P<0.05, ††P<0.05 vs the PA group.
3. 讨 论
Ansermet等[12]研究表明：在足细胞中特异敲除昼夜节律系统的关键基因BAML1导致足细胞的功能受损，肾小球的滤过率下降，尿肌酐、钠、钾及水的排泄受损。分子机制可能为敲除BAML1导致足细胞功能受损的标志基因的昼夜节律表达消失，参与足细胞分化、代谢、细胞骨架形成及黏附功能的基因表达失调，如Tcf21、解偶联蛋白1(uncoupling protein 2，Ucp2)等。
研究[8-11]显示：昼夜节律系统基因参与脂质代谢的调控，其中PER3负调控脂肪细胞的分化，减少脂质的合成。分子机制研究[8-9]表明：PER3能与脂肪分化关键的核转录因子PPARγ相互作用并下调PPARγ的表达。此外，PER3还能与转录因子Klf15结合下调其表达并抑制脂肪细胞分化[10-11]。而Klf15的下调也有可能导致足细胞的炎症增加，并影响足细胞骨架的形成及足细胞的分化等[13-14]。为了探讨PER3在保护高脂诱导的足细胞损伤中的作用机制，作者首先检索了Pubmed中的GEO profiles数据库，发现PER3在肥胖患者脂肪干细胞及脂肪细胞中的表达较正常体重人群显著下调。为了检测PER3在肥胖与正常体重小鼠肾中的表达，作者利用高脂饮食诱导肥胖小鼠模型，发现在肥胖小鼠肾中PER3蛋白表达较在正常体重小鼠肾中的表达显著下调，表明PER3在肥胖导致的肾损害中可能扮演重要角色。随后利用不同浓度PA干预足细胞，发现150 μmol/L PA导致PER3 mRNA及蛋白质的表达显著下调，并随着干预时间的延长PER3 mRNA及蛋白质的表达也逐渐下降。以上结果表明PER3可能负调控PA诱导的足细胞中的脂质合成。为了进一步验证推测，作者在足细胞中过表达及沉默PER3，发现过表达PER3能减少PA诱导的足细胞内TG的形成，而沉默PER3则相反。同时还发现：过表达PER3能显著增加PA诱导的足细胞标志基因的下调，如nephrin、podocin、podocalyxin及podoplanin。
为了进一步探讨PER3在PA诱导的足细胞损伤中的保护机制，作者在足细胞中沉默PER3，利用RNA-seq测序检测了与对照组的差异基因表达，通路分析发现差异表达基因在氧化磷酸化、TNF信号通路及ECM受体的相互作用通路中显著富集，且脂质代谢相关通路(如游离脂肪酸代谢通路、游离脂肪酸降解通路)中也有部分富集，表明PER3在足细胞的脂质代谢中起关键作用。而氧化应激因子、炎症因子及ECM的增多均是导致足细胞功能受损的重要原因，ECM的增多导致肾小球基底膜增厚，随后导致局灶节段性肾小球硬化[15-18]。通络分析及GO分析为寻找PER3在足细胞PA损伤中的保护机制提供了依据。
细胞内氧化应激及炎症因子的增加是高脂诱导足细胞受损的关键原因。尽管适当的ROS为足细胞增殖和分化所必须，但过多的细胞内脂质的累积导致线粒体电子链的传递受损，ROS的清除下降，引起足细胞内蛋白质的氧化、脂质过氧化、DNA的破坏，导致足细胞的功能受损及凋亡[19-21]。足细胞对PA诱导的氧化应激非常敏感，在PA暴露的早期即开始启动凋亡的程序[21]，同时，ROS及氧化的高密度及低密度脂蛋白均可激活MAPK、NF-κB等炎症因子通路，导致炎症细胞浸润以及IL-6、TNF-α等炎症因子的分泌增加[22-23]。为了验证PER3对PA诱导的足细胞氧化应激及炎症的保护作用，本研究发现PA诱导足细胞SOD1、GXP1及CAT mRNA表达的下调，而过表达PER3可显著上调这些基因的表达；此外，PA可诱导足细胞内MDA、TNF-α、IL-6、IL-1β及IL-2含量增加，而过表达PER3减少这些炎症及氧化应激因子的含量，GSH则相反。本研究结果还证明PER3能减少足细胞中PA诱导的氧化应激及炎症因子的分泌。
综上所述，昼夜节律调控因子PER3能抑制PA诱导的足细胞内脂质合成，减少PA诱导的足细胞氧化应激及炎症因子的产生，本研究为昼夜节律调控基因参与高脂诱导的足细胞的保护提供了新的思路及治疗的靶点。
基金资助
国家自然科学基金(81870589)。
This work was supported by the National Natural Science Foundation of China (81870589).
利益冲突声明
作者声称无任何利益冲突。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/2021111177.pdf
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